Expansion of definitive endoderm cells

ABSTRACT

Disclosed herein are cell cultures comprising expanded definitive endoderm cells as well as methods for expanding definitive endoderm cells in culture.

RELATED APPLICATIONS

This application is a division of and claims priority to U.S. patentapplication Ser. No. 11/317,387, entitled EXPANSION OF DEFINITIVEENDODERM CELLS, filed Dec. 22, 2005, which is a continuation-in-part ofand claims priority to U.S. patent application Ser. No. 11/021,618,entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, now U.S. Pat. No.7,510,876, issued Mar. 31, 2009, which claims priority under 35 U.S.C.§119(e) as a nonprovisional application to U.S. Provisional PatentApplication No. 60/587,942, entitled CHEMOKINE CELL SURFACE RECEPTOR FORTHE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 14, 2004, U.S.Provisional Patent Application No. 60/586,566, entitled CHEMOKINE CELLSURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9,2004 and U.S. Provisional Patent Application No. 60/532,004, entitledDEFINITIVE ENDODERM, filed Dec. 23, 2003; U.S. patent application Ser.No. 11/317,387 is also a nonprovisional application of and claimspriority to U.S. Provisional Patent Application No. 60/693,317, entitledEXPANSION OF ISOLATED DEFINITIVE ENDODERM CELLS, filed Jun. 23, 2005 anda nonprovisional application of and claims priority to U.S. ProvisionalPatent Application No. 60/736,598, entitled MARKERS OF DEFINITIVEENDODERM, filed Nov. 14, 2005.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and cellbiology. In particular, the present invention relates to compositions ofdefinitive endoderm cells which have been expanded either prior to orsubsequent to enrichment, isolation and/or purification as well asmethods of producing and using such cells.

BACKGROUND

Human pluripotent stem cells, such as embryonic stem (ES) cells andembryonic germ (EG) cells, were first isolated in culture withoutfibroblast feeders in 1994 (Bongso et al., 1994) and with fibroblastfeeders (Hogan, 1997). Later, Thomson, Reubinoff and Shamblottestablished continuous cultures of human ES and EG cells usingmitotically inactivated mouse feeder layers (Reubinoff et al., 2000;Shamblott et al., 1998; Thomson et al., 1998).

Human ES and EG cells (hESCs) offer unique opportunities forinvestigating early stages of human development as well as fortherapeutic intervention in several disease states, such as diabetesmellitus and Parkinson's disease. For example, the use ofinsulin-producing β-cells derived from hESCs would offer a vastimprovement over current cell therapy procedures that utilize cells fromdonor pancreases for the treatment of diabetes. However, presently it isnot known how to generate an insulin-producing β-cell from hESCs. Assuch, current cell therapy treatments for diabetes mellitus, whichutilize islet cells from donor pancreases, are limited by the scarcityof high quality islet cells needed for transplant. Cell therapy for asingle Type I diabetic patient requires a transplant of approximately8×10⁸ pancreatic islet cells. (Shapiro et al., 2000; Shapiro et al.,2001a; Shapiro et al., 2001b). As such, at least two healthy donororgans are required to obtain sufficient islet cells for a successfultransplant. Human embryonic stem cells offer a source of startingmaterial from which to develop substantial quantities of high qualitydifferentiated cells for human cell therapies.

Two properties that make hESCs uniquely suited to cell therapyapplications are pluripotence and the ability to maintain these cells inculture for prolonged periods. Pluripotency is defined by the ability ofhESCs to differentiate to derivatives of all 3 primary germ layers(endoderm, mesoderm, ectoderm) which, in turn, form all somatic celltypes of the mature organism in addition to extraembryonic tissues (e.g.placenta) and germ cells. Although pluripotency imparts extraordinaryutility upon hESCs, this property also poses unique challenges for thestudy and manipulation of these cells and their derivatives. Owing tothe large variety of cell types that may arise in differentiating hESCcultures, the vast majority of cell types are produced at very lowefficiencies in mixed cell populations. In order to use hESCs as astarting material to generate cells that are useful in cell therapyapplications, it would be advantageous to overcome the foregoingproblems.

SUMMARY OF THE INVENTION

Embodiments of the present invention relate to cell cultures comprisingexpanded definitive endoderm cells. Further embodiments described hereinrelate to methods for expanding enriched, isolated and/or purifieddefinitive endoderm cells in culture.

Some of the methods described herein relate to the maintenance, growth,passage and/or expansion of definitive endoderm cells in cell culture.In such embodiments, cell cultures comprising definitive endoderm cellsare obtained. The cells are then isolated so that at least some of thedefinitive endoderm cells are separated from at least some of the othercells in the cell culture, thereby producing a cell population that isenriched in definitive endoderm cells. In some embodiments, the enrichedcell populations of definitive endoderm cells are cultured underconditions that permit the expansion of the definitive endoderm cells.

In other embodiments of the methods described herein, the definitiveendoderm cells are multipotent cells that can differentiate into cellsof the gut tube or organs derived therefrom. In preferred embodiments,the definitive endoderm cells are human definitive endoderm cells thatare obtained by differentiating human embryonic stem cells (hESCs). Insuch embodiments, definitive endoderm cells can be derived from hESCs bycontacting such cells with at least one growth factor from the TGFβsuperfamily, such as activin A. In other embodiments, human and/or otherdefinitive endoderm cells can be obtained from a pre-existing culture ofdefinitive endoderm cells. In such embodiments, either a portion of orthe entire culture may be used in the definitive endoderm expansionmethods described herein.

In addition to obtaining cell cultures comprising definitive endodermcells, some embodiments of the expansion methods described herein alsocomprise the step of producing enriched definitive endoderm cellpopulations. In some embodiments, such enriched definitive endoderm cellpopulations are produced by separating at least some of the definitiveendoderm cells from at least some of the other cells in the cellcultures. As such, the at least some of the definitive endoderm cellsare isolated from at least some of the other cells which remain in thecell culture. In some embodiments, the isolating step comprisesproviding the cells in the cell culture with a reagent which binds to amarker expressed in said definitive endoderm cells but which is notsubstantially expressed in said other cells present in the cell culture.The reagent-bound definitive endoderm cells are then separated from thenon-reagent-bound cells, thereby producing an enriched definitiveendoderm cell population. In some embodiments, the marker is CXCR4 andthe reagent is an antibody with an affinity for CXCR4. In someembodiments, the definitive endoderm cells are separated by fluorescenceactivated cell sorting (FACS).

In still other embodiments, at least some of the definitive endodermcells are separated from at least some of the other cells in the cultureby specifically fluorescently labeling the definitive endoderm cells inculture and then separating the labeled cells from the unlabeled cellsby FACS. In some embodiments, the fluorescence is produced by greenfluorescent protein (GFP) or enhanced green fluorescent protein (EGFP).In some embodiments, the GFP and/or EGFP is expressed under the controlof the SOX17 or the CXCR4 promoter.

In some embodiments, the enriched definitive endoderm cell populationsthat are produced as described above are substantially free of cellsother than definitive endoderm cells. In other embodiments, the enricheddefinitive endoderrn cell populations comprise from at least about 96%to at least about 100% definitive endoderm cells.

Additional embodiments of the methods described herein also include aculturing step that comprises plating the population enriched indefinitive endoderm cells or a portion of the population. In someembodiments, the cells are plated on a surface coated with humanfibronectin and/or poly-ornithine. In other embodiments, the culturingstep comprises incubating the enriched definitive endoderm cellpopulation or portion thereof in a medium comprising about 2% (v/v)serum. In some embodiments, the medium also comprises at least onegrowth factor. In certain embodiments, the at least one growth factor isa growth factor comprises a member of the TGFβ superfamily, such asactivin A. Alternatively, the growth factor can be IGF1, bFGF, EGF oranother growth factor. In such embodiments, the growth factor can bepresent in the medium at a concentration ranging from about 1 ng/ml toabout 5000 ng/ml. In some embodiments, a combination of growth factorsis present in the culture medium.

Additional embodiments described herein relate to methods of expandingdefinitive endoderm cells in culture by obtaining a cell culturecomprising definitive endoderm cells and then passaging the definitiveendoderm cells so as to produce a plurality of cell cultures comprisingdefinitive endoderm cells. In some embodiments, the definitive endodermcells obtained in cell culture are attached to a substrate, such as thesurface of a cell culture flask or the surface of a microtiter plate. Insome embodiments, the definitive endoderm cells are passaged usingenzymatic methods. In other embodiments, the definitive endoderm cellsare mechanically passaged. In yet other embodiments, the definitiveendoderm cells are passage using a cell dispersal buffer.

Still other embodiments described herein relate to expanded definitiveendoderm cell cultures and/or populations produced by the methodsdescribed herein. In such embodiments, the definitive endoderm cells aremultipotent cells that can differentiate into cells of the gut tube ororgans derived therefrom.

In certain jurisdictions, there may not be any generally accepteddefinition of the term “comprising.” As used herein, the term“comprising” is intended to represent “open” language which permits theinclusion of any additional elements. With this in mind, additionalembodiments of the present inventions are described with reference tothe numbered paragraphs below:

1. A method of expanding definitive endoderm cells in culture, saidmethod comprising the steps of: (a) obtaining a cell culture comprisingdefinitive endoderm cells, (b) isolating at least some of the definitiveendoderm cells from at least some of the other cells in the cellculture, thereby producing a cell population enriched in definitiveendoderm cells; and (c) culturing said cell population enriched indefinitive endoderm cells under conditions that permit the expansion ofsaid definitive endoderm cells.

2. The method of paragraph 1, wherein said definitive endoderm cellsbeing multipotent cells that can differentiate into cells of the guttube or organs derived therefrom.

3. The method of paragraph 1, wherein said definitive endoderm cells arehuman definitive endoderm cells.

4. The method of paragraph 3, wherein said definitive endoderm cells arederived from human embryonic stem cells (hESCs).

5. The method of paragraph 4, wherein the obtaining step comprisescontacting hESCs with at least one growth factor from the TGFβsuperfamily so as to permit differentiation of at least some of saidhESCs to definitive endoderm cells.

6. The method of paragraph 5, wherein said at least one growth factorfrom the TGFβ superfamily comprises activin A.

7. The method of paragraph 1, wherein the step of obtaining said cellculture comprising definitive endoderm further comprises obtaining aportion of an existing definitive endoderm culture.

8. The method of paragraph 1, wherein said cell population enriched indefinitive endoderm cells is substantially free of cells other thandefinitive endoderm cells.

9. The method of paragraph 1, wherein said cell population enriched indefinitive endoderm cells comprises at least about 96% definitiveendoderm cells.

10. The method of paragraph 1, wherein said cell population enriched indefinitive endoderm cells comprises at least about 97% definitiveendoderm cells.

11. The method of paragraph 1, wherein said cell population enriched indefinitive endoderm cells comprises at least about 98% definitiveendoderm cells.

12. The method of paragraph 1, wherein said cell population enriched indefinitive endoderm cells comprises at least about 99% definitiveendoderm cells.

13. The method of paragraph 1, wherein said cell population enriched indefinitive endoderm cells comprises about 100% definitive endodermcells.

14. The method of paragraph 1, wherein said isolating step comprisesproviding said cell culture with a reagent which binds to a markerexpressed in said definitive endoderm cells but which is notsubstantially expressed in said other cells present in the cell culture,and separating said definitive endoderm cells bound to said reagent fromsaid other cells present in the cell culture, thereby producing a cellpopulation enriched in definitive endoderm cells.

15. The method of paragraph 14, wherein said marker is CXCR4.

16. The method of paragraph 14, wherein said reagent is an antibody.

17. The method of paragraph 16, wherein said antibody has affinity forCXCR4.

18. The method of paragraph 14, wherein said definitive endoderm cellsbound to said reagent are separated from said other cells present in thecell culture by fluorescence activated cell sorting (FACS).

19. The method of paragraph 1, wherein said isolating step comprisesseparating fluorescently-labeled definitive endoderm cells fromunlabeled cells.

20. The method of paragraph 19, wherein said fluorescently-labeleddefinitive endoderm cells are labeled as a result of the expression ofenhanced green fluorescent protein (EGFP).

21. The method of paragraph 20, wherein the expression of EGFP is undercontrol of the SOX17 promoter.

22. The method of paragraph 20, wherein the expression of EGFP is undercontrol of the CXCR4 promoter.

23. The method of paragraph 19, wherein said fluorescently-labeleddefinitive endoderm cells are separated from unlabeled cells by FACS.

24. The method of paragraph 1, wherein said culturing step comprisesplating said population enriched in definitive endoderm cells or aportion thereof.

25. The method of paragraph 24, wherein said population enriched indefinitive endoderm cells or a portion thereof is plated on a surfacecoated with human fibronectin.

26. The method of paragraph 25, wherein said surface is coated withpoly-ornithine.

27. The method of paragraph 1, wherein said culturing step comprisesincubating said population enriched in definitive endoderm cells or aportion thereof in a medium comprising about 2% (v/v) serum.

28. The method of paragraph 1, wherein said culturing step comprisesincubating said population enriched in definitive endoderm cells or aportion thereof in a medium comprising greater than about 2% (v/v)serum.

29. The method of paragraph 1, wherein said culturing step comprisesincubating said population enriched in definitive endoderm cells or aportion thereof in a medium comprising less than about 2% (v/v) serum.

30. The method of paragraph 1, wherein said culturing step comprisesincubating said population enriched in definitive endoderm cells or aportion thereof in a medium comprising at least one growth factor.

31 The method of paragraph 30, wherein said at least one growth factoris a growth factor from the TGFβ superfamily of growth factors.

32. The method of paragraph 31, wherein said at least one growth factorfrom the TGFβ superfamily of growth factors comprises activin A. 33. Themethod of paragraph 32, wherein said activin A is present in said mediumat a concentration of about 100 ng/ml.

34. The method of paragraph 30, wherein said at least one growth factorcomprises IGF1.

35. The method of paragraph 34, wherein said IGF1 is present in saidmedium at a concentration of about 100 ng/ml.

36. The method of paragraph 30, wherein said at least one growth factorcomprises a combination of activin A and IGF1.

37. The method of paragraph 30, wherein said at least one growth factorcomprises bFGF.

38. The method of paragraph 37, wherein said bFGF is present in saidmedium at a concentration of about 12 ng/ml.

39. The method of paragraph 30, wherein said at least one growth factorcomprises EGF.

40. The method of paragraph 39, wherein said EGF is present in saidmedium at a concentration of about 10 ng/ml.

41. The method of paragraph 30, wherein said at least one growth factorcomprises a combination of activin A, bFGF and EGF.

42. An expanded definitive endoderm cell population produced by themethod of paragraph 1.

43. A method of expanding definitive endoderm cells in culture, saidmethod comprising the steps of: (a) obtaining a cell culture comprisingdefinitive endoderm cells, and (b) passaging said definitive endodermcells, thereby producing a plurality of cell cultures comprisingdefinitive endoderm cells.

44. The method of paragraph 43, wherein the step of passaging saiddefinitive endoderm cells comprises providing at least one enzyme tosaid cell culture.

45. The method of paragraph 44, wherein said at least one enzymecomprises at least one protease.

46. The method of paragraph 45, wherein said at least one proteasecomprises trypsin.

47. The method of paragraph 43, wherein the step of passaging saiddefinitive endoderm cells comprises mechanically disrupting contactsbetween said definitive endoderm cells.

48. The method of paragraph 43, wherein the step of passaging saiddefinitive endoderm cells comprises incubating said definitive endodermcells in a cell dispersal buffer.

49. The method of paragraph 43, wherein said definitive endoderm cellsare attached to a substrate.

50. The method of paragraph 49, wherein the step of passaging saiddefinitive endoderm cells comprises detaching said definitive endodermcells from said substrate.

51. The method of paragraph 50, wherein said substrate is a surface of atissue culture flask.

52. The method of paragraph 50, wherein said substrate is a surface of amicrotiter plate.

53. An expanded definitive endoderm cell population produced by themethod of paragraph 43.

It will be appreciated that the methods and compositions described aboverelate to cells cultured in vitro. However, the above-described in vitrodifferentiated cell compositions may be used for in vivo applications.

Additional embodiments of the present invention may also be found inU.S. Provisional Patent Application No. 60/532,004, entitled DEFINITIVEENDODERM, filed Dec. 23, 2003; U.S. Provisional Patent Application No.60/566,293, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 27, 2004; U.S.Provisional Patent Application No. 60/586,566, entitled CHEMOKINE CELLSURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9,2004; U.S. Provisional Patent Application No. 60/587,942, entitledCHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVEENDODERM, filed Jul. 14, 2004; U.S. patent application Ser. No.11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004; U.S.patent application Ser. No. 11/115,868, entitled PDX1 EXPRESSINGENDODERM, filed Apr. 26, 2005; U.S. patent application Ser. No.11/165,305, entitled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATINGDEFINITIVE ENDODERM, filed Jun. 23, 2005; U.S. Provisional PatentApplication No. 60/693364, entitled PREPRIMITIVE STREAK AND MESENDODERMCELLS, filed Jun. 23, 2005; U.S. Provisional Patent Application No.60/693,317, entitled EXPANSION OF ISOLATED DEFINITIVE ENDODERM CELLS,filed Jun. 23, 2005; and U.S. Provisional Patent Application No.60/736,598, entitled MARKERS OF DEFINITIVE ENDODERM, filed Nov. 14,2005, the disclosures of which are incorporated herein by reference intheir entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a proposed differentiation pathway for theproduction of beta-cells from hESCs. The first step in the pathwaycommits the ES cell to the definitive endoderm lineage and representsone of the earliest known steps in the further differentiation of EScells to pancreatic endoderm, endocrine endoderm, or islet/beta-cell.Some factors useful for mediating this transition are members of theTGFβ family which include, but are not limited to, activins and nodals.Exemplary markers for defining the definitive endoderm target cell areSOX17, GATA4, HNF3b, MIX1 and CXCR4.

FIG. 2 is a diagram of the human SOX17 cDNA which displays the positionsof conserved motifs and highlights the region used for the immunizationprocedure by GENOVAC.

FIG. 3 is a relational dendrogram illustrating that SOX17 is mostclosely related to SOX7 and somewhat less to SOX18. The SOX17 proteinsare more closely related among species homologs than to other members ofthe SOX group F subfamily within the same species.

FIG. 4 is a Western blot probed with the rat anti-SOX17 antibody. Thisblot demonstrates the specificity of this antibody for human SOX17protein over-expressed in fibroblasts (lane 1) and a lack ofimmunoreactivity with EGFP (lane 2) or the most closely related SOXfamily member, SOX7 (lane 3).

FIGS. 5A-B are micrographs showing a cluster of SOX17⁺ cells thatdisplay a significant number of AFP⁺ co-labeled cells (A). This is instriking contrast to other SOX17⁺ clusters (B) where little or no AFP⁺cells are observed.

FIGS. 6A-C are micrographs showing parietal endoderm and SOX17. Panel Ashows immunocytochemistry for human Thrombomodulin (TM) protein locatedon the cell surface of parietal endoderm cells in randomlydifferentiated cultures of hES cells. Panel B is the identical fieldshown in A double-labeled for TM and SOX17. Panel C is the phasecontrast image of the same field with DAPI labeled nuclei. Note thecomplete correlation of DAPI labeled nuclei and SOX17 labeling.

FIGS. 7A-B are bar charts showing SOX17 gene expression by quantitativePCR (Q-PCR) and anti-SOX17 positive cells by SOX17-specific antibody.Panel A shows that activin A increases SOX17 gene expression whileretinoic acid (RA) strongly suppresses SOX17 expression relative to theundifferentiated control media (SR20). Panel B shows the identicalpattern as well as a similar magnitude of these changes is reflected inSOX17⁺ cell number, indicating that Q-PCR measurement of SOX17 geneexpression is very reflective of changes at the single cell level.

FIG. 8A is a bar chart which shows that a culture of differentiatinghESCs in the presence of activin A maintains a low level of AFP geneexpression while cells allowed to randomly differentiate in 10% fetalbovine serum (FBS) exhibit a strong upregulation of AFP. The differencein expression levels is approximately 7-fold.

FIGS. 8B-C are images of two micrographs showing that the suppression ofAFP expression by activin A is also evident at the single cell level asindicated by the very rare and small clusters of AFP⁺ cells observed inactivin A treatment conditions (bottom) relative to 10% FBS alone (top).

FIGS. 9A-B are comparative images showing the quantitation of the AFP⁺cell number using flow cytometry. This figure demonstrates that themagnitude of change in AFP gene expression (FIG. 8A) in the presence(right panel) and absence (left panel) of activin A exactly correspondsto the number of AFP⁺ cells, further supporting the utility of Q-PCRanalyses to indicate changes occurring at the individual cell level.

FIGS. 10A-F are micrographs which show that exposure of hESCs to nodal,activin A and activin B (NAA) yields a striking increase in the numberof SOX17⁺ cells over the period of 5 days (A-C). By comparing to therelative abundance of SOX17⁺ cells to the total number of cells presentin each field, as indicated by DAPI stained nuclei (D-F), it can be seenthat approximately 30-50% of all cells are immunoreactive for SOX17after five days treatment with NAA.

FIG. 11 is a bar chart which demonstrates that activin A (0, 10, 30 or100 ng/ml) dose-dependently increases SOX17 gene expression indifferentiating hESCs. Increased expression is already robust after 3days of treatment on adherent cultures and continues through subsequent1, 3 and 5 days of suspension culture as well.

FIGS. 12A-C are bar charts which demonstrate the effect of activin A onthe expression of MIXL1 (panel A), GATA4 (panel B) and HNF3b (panel C).Activin A dose-dependent increases are also observed for three othermarkers of definitive endoderm; MIXL1, GATA4 and HNf3b. The magnitudesof increased expression in response to activin dose are strikinglysimilar to those observed for SOX17, strongly indicating that activin Ais specifying a population of cells that co-express all four genes(SOX17⁺, MIXL1⁺, GATA4⁺ and HNF3b⁺).

FIGS. 13A-C are bar charts which demonstrate the effect of activin A onthe expression of AFP (panel A), SOX7 (panel B) and SPARC (panel C).There is an activin A dose-dependent decrease in expression of thevisceral endoderm marker AFP. Markers of primitive endoderm (SOX7) andparietal endoderm (SPARC) remain either unchanged or exhibit suppressionat some time points indicating that activin A does not act to specifythese extra-embryonic endoderm cell types. This further supports thefact that the increased expression of SOX17, MIXL1, GATA4, and HNF3b aredue to an increase in the number of definitive endoderm cells inresponse to activin A.

FIGS. 14A-B are bar charts showing the effect of activin A on ZIC1(panel A) and Brachyury expression (panel B) Consistent expression ofthe neural marker ZIC1 demonstrates that there is not a dose-dependenteffect of activin A on neural differentiation. There is a notablesuppression of mesoderm differentiation mediated by 100 ng/ml of activinA treatment as indicated by the decreased expression of brachyury. Thisis likely the result of the increased specification of definitiveendoderm from the mesendoderm precursors. Lower levels of activin Atreatment (10 and 30 ng/ml) maintain the expression of brachyury atlater time points of differentiation relative to untreated controlcultures.

FIGS. 15A-B are micrographs showing decreased parietal endodermdifferentiation in response to treatment with activins. Regions ofTM^(hi) parietal endoderm are found through the culture (A) whendifferentiated in serum alone, while differentiation to TM⁺ cells isscarce when activins are included (B) and overall intensity of TMimmunoreactivity is lower.

FIGS. 16A-D are micrographs which show marker expression in response totreatment with activin A and activin B. hESCs were treated for fourconsecutive days with activin A and activin B and triple labeled withSOX17, AFP and TM antibodies. Panel A-SOX17; Panel B-AFP; Panel C-TM;and Panel D-Phase/DAPI. Notice the numerous SOX17 positive cells (A)associated with the complete absence of AFP (B) and TM (C)immunoreactivity.

FIG. 17 is a micrograph showing the appearance of definitive endodermand visceral endoderm in vitro from hESCs. The regions of visceralendoderm are identified by AFP^(hi)/SOX17^(lo/−) while definitiveendoderm displays the complete opposite profile, SOX17^(hi)/AFP^(lo/−).This field was selectively chosen due to the proximity of these tworegions to each other. However, there are numerous times whenSOX17^(hi)/AFP^(lo/−) regions are observed in absolute isolation fromany regions of AFP^(hi) cells, suggesting the separate origination ofthe definitive endoderm cells from visceral endoderm cells.

FIG. 18 is a diagram depicting the TGFβ family of ligands and receptors.Factors activating AR Smads and BR Smads are useful in the production ofdefinitive endoderm from human embryonic stem cells (see, J CellPhysiol. 187:265-76).

FIG. 19 is a bar chart showing the induction of SOX17 expression overtime as a result of treatment with individual and combinations of TGFβfactors.

FIG. 20 is a bar chart showing the increase in SOX17⁺ cell number withtime as a result of treatment with combinations of TGFβ factors.

FIG. 21 is a bar chart showing induction of SOX17 expression over timeas a result of treatment with combinations of TGFβ factors.

FIG. 22 is a bar chart showing that activin A induces a dose-dependentincrease in SOX17⁺ cell number.

FIG. 23 is a bar chart showing that addition of Wnt3a to activin A andactivin B treated cultures increases SOX17 expression above the levelsinduced by activin A and activin B alone.

FIGS. 24A-C are bar charts showing differentiation to definitiveendoderm is enhanced in low FBS conditions. Treatment of hESCs withactivins A and B in media containing 2% FBS (2AA) yields a 2-3 timesgreater level of SOX17 expression as compared to the same treatment in10% FBS media (10AA) (panel A). Induction of the definitive endodermmarker MIXL1 (panel B) is also affected in the same way and thesuppression of AFP (visceral endoderm) (panel C) is greater in 2% FBSthan in 10% FBS conditions.

FIGS. 25A-D are micrographs which show SOX17⁺ cells are dividing inculture. SOX17 immunoreactive cells are present at the differentiatingedge of an HESC colony (C, D) and are labeled with proliferating cellnuclear antigen (PCNA) (panel B) yet are not co-labeled with OCT4 (panelC). In addition, clear mitotic figures can be seen by DAPI labeling ofnuclei in both SOX17⁺ cells (arrows) as well as OCT4⁺, undifferentiatedhESCs (arrowheads) (D).

FIG. 26 is a bar chart showing the relative expression level of CXCR4 indifferentiating hESCs under various media conditions. FIGS. 27A-D arebar charts that show how a panel of definitive endoderm markers share avery similar pattern of expression to CXCR4 across the samedifferentiation treatments displayed in FIG. 26.

FIGS. 28A-E are bar charts showing how markers for mesoderm (BRACHYURY,MOX1), ectoderm (SOX1, ZIC1) and visceral endoderm (SOX7) exhibit aninverse relationship to CXCR4 expression across the same treatmentsdisplayed in FIG. 26.

FIGS. 29A-F are micrographs that show the relative difference in SOX17immunoreactive cells across three of the media conditions displayed inFIGS. 26-28.

FIGS. 30A-C are flow cytometry dot plots that demonstrate the increasein CXCR4⁺ cell number with increasing concentration of activin A addedto the differentiation media.

FIGS. 31A-D are bar charts that show the CXCR4⁺ cells isolated from thehigh dose activin A treatment (A100-CX+) are even further enriched fordefinitive endoderm markers than the parent population (A100).

FIG. 32 is a bar chart showing gene expression from CXCR4⁺ and CXCR4⁻cells isolated using fluorescence-activated cell sorting (FACS) as wellas gene expression in the parent populations. This demonstrates that theCXCR4⁺ cells contain essentially all the CXCR4 gene expression presentin each parent population and the CXCR4⁻ populations contain very littleor no CXCR4 gene expression.

FIGS. 33A-D are bar charts that demonstrate the depletion of mesoderm(BRACHYURY, MOX1), ectoderm (ZIC1) and visceral endoderm (SOX7) geneexpression in the CXCR4+ cells isolated from the high dose activin Atreatment which is already suppressed in expression of thesenon-definitive endoderm markers.

FIGS. 34A-M are bar charts showing the expression patterns of markergenes that can be used to identify definitive endoderm cells. Theexpression analysis of definitive endoderm markers, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1 is shown in panels G-L, respectively. Theexpression analysis of previously described lineage marking genes,SOX17, SOX7, SOX17/SQX7, TM, ZIC1, and MOX1 is shown in panels A-F,respectively. Panel M shows the expression analysis of CXCR4. Withrespect to each of panels A-M, the column labeled hESC indicates geneexpression from purified human embryonic stem cells; 2NF indicates cellstreated with 2% FBS, no activin addition; 0.1A100 indicates cellstreated with 0.1% FBS, 100 ng/ml activin A; 1A100 indicates cellstreated with 1% FBS, 100 ng/ml activin A; and 2A100 indicates cellstreated with 2% FBS, 100 ng/ml activin A.

FIGS. 35A-D are bar charts showing the expression patterns of definitiveendoderm marker genes in cell cultures maintained for 36 days undervarious growth conditions. The expression analysis of definitiveendoderm markers SOX17, GSC, MIXL1 and CXCR4 is shown in panels A-D,respectively. EB and EV are used to designate to separate cellpopulations each from the hCyT25 hESC line. The abbreviation NFindicates cells grown in the absence of activin A, whereas A100indicates cells grown in the presence of 100 ng/ml this factor. EGFindicates 50 ng/ml epidermal growth factor.

FIG. 36 is a diagram showing the cell differentiation, isolation andexpansion procedure for definitive endoderm cells. The abbreviations areas follows: hESC refers to human embryonic stem cells; d5 hESC-DE refersto unpurified definitive endoderm cells; d6 FACS-DE refers to CXCR4antibody/FACS purified definitive endoderm cells; and p1 d10 FACS-DErefers to purified definitive endoderm cells that have been passagedonce and grown for 10 additional days subsequent to passage. RNA samplesare taken and analyzed for marker expression at each of the indicateddays.

FIGS. 37A-F are bar charts showing the expression patterns of variousembryonic cell lineage marker genes in cell cultures that have beendifferentiated to definitive endoderm then subsequently purified usingthe CXCR4 antibody in conjunction with fluorescence activated cellsorting (FACS). The abbreviations are as follows: p96 hESC refers tomRNA from the 96^(th) passage of CyT25 human embryonic stem cells; d5 DErefers to mRNA from unpurified definitive endoderm cells on the fifthday of differentiation from p96 hESCs; NF d6-FACS refers to mRNA taken11 days post differentiation from CXCR4 antibody/FACS purifieddefinitive endoderm cells incubated in the absence of activin A; Arefers to mRNA taken 11 days post differentiation from CXCR4antibody/FACS purified definitive endoderm cells incubated in thepresence of 100 ng/ml activin A; Al refers to mRNA taken 11 days postdifferentiation from CXCR4 antibody/FACS purified definitive endodermcells incubated in the presence of 100 ng/ml activin A and 100 ng/mlIGF1; AFE refers to mRNA taken 11 days post differentiation from CXCR4antibody/FACS purified definitive endoderm cells incubated in thepresence of 100 ng/ml activin A, 12 ng/ml bFGF and 10 ng/ml EGF; NFp1-d10-FACS refers to mRNA taken 10 days post passage of CXCR4antibody/FACS purified definitive endoderm cells incubated in theabsence of activin A; A refers to mRNA taken 10 days post passage fromCXCR4 antibody/FACS purified definitive endoderm cells incubated in thepresence of 100 ng/ml activin A; AI refers to mRNA taken 10 days postpassage from CXCR4 antibody/FACS purified definitive endoderm cellsincubated in the presence of 100 ng/ml activin A and 100 ng/ml IGF1; andAFE refers to mRNA taken 10 days post passage from CXCR4 antibody/FACSpurified definitive endoderm cells incubated in the presence of 100ng/ml activin A, 12 ng/ml bFGF and 10 ng/ml EGF. Panel A-SOX17; B-GSC;C-OCT4; D-Brachyury; E-ZIC1; F-SOX1.

DETAILED DESCRIPTION

A crucial stage in early human development termed gastrulation occurs2-3 weeks after fertilization. Gastrulation is extremely significantbecause it is at this time that the three primary germ layers are firstspecified and organized (Lu et al., 2001; Schoenwolf and Smith, 2000).The ectoderm is responsible for the eventual formation of the outercoverings of the body and the entire nervous system whereas the heart,blood, bone, skeletal muscle and other connective tissues are derivedfrom the mesoderm. Definitive endoderm is defined as the germ layer thatis responsible for formation of the entire gut tube which includes theesophagus, stomach and small and large intestines, and the organs whichderive from the gut tube such as the lungs, liver, thymus, parathyroidand thyroid glands, gall bladder and pancreas (Grapin-Botton and Melton,2000; Kimelman and Griffin, 2000; Tremblay et al., 2000; Wells andMelton, 1999; Wells and Melton, 2000). A very important distinctionshould be made between the definitive endoderm and the completelyseparate lineage of cells termed primitive endoderm. The primitiveendoderm is primarily responsible for formation of extra-embryonictissues, mainly the parietal and visceral endoderm portions of theplacental yolk sac and the extracellular matrix material of Reichert'smembrane.

During gastrulation, the process of definitive endoderm formation beginswith a cellular migration event in which mesendoderm cells (cellscompetent to form mesoderm or endoderm) migrate through a structurecalled the primitive streak. Definitive endoderm is derived from cells,which migrate through the anterior portion of the streak and through thenode (a specialized structure at the anterior-most region of thestreak). As migration occurs, definitive endoderm populates first themost anterior gut tube and culminates with the formation of theposterior end of the gut tube.

Definitive endoderm and endoderm cells derived therefrom representimportant multipotent starting points for the derivation of cells whichmake up terminally differentiated tissues and/or organs derived from thedefinitive endoderm lineage. Such cells, tissues and/or organs areextremely useful in cell therapies. Because large numbers of cells areusually necessary for successful cell therapy applications, it isbeneficial to begin differentiation procedures with large numbers ofcells of a single cell type. As embryonic stem cells in culturedifferentiate to definitive endoderm, not every embryonic stem cell isconverted to the definitive endoderm cell type. To overcome thisproblem, definitive endoderm cells growing in mixed cell cultures can beenriched, isolated and/or purified using the methodology describedherein. After such enrichment, isolation and/or purification, theresulting definitive endoderm cells may not be easy to grow in culture.Methods described herein improve the ability of enriched, isolatedand/or purified definitive endoderm cells to grow and expand in cellculture. Because definitive endoderm cells can now be expanded inculture subsequent to enrichment, isolation and/or purification, cells,tissues and/or organs derived from definitive endoderm cells can beproduced in greater numbers.

Some embodiments of the present invention relate to methods of expandingdefinitive endoderm cells in cell culture. In some embodiments,definitive endoderm cells are enriched by separating these cells fromother cells in a mixed cell culture. The enriched definitive endodermcells are then cultured under conditions which permit their expansion.

Definitions

Certain terms and phrases as used throughout this application have themeanings provided as follows:

As used herein, “embryonic” refers to a range of developmental stages ofan organism beginning with a single zygote and ending with amulticellular structure that no longer comprises pluripotent ortotipotent cells other than developed gametic cells. In addition toembryos derived by gamete fusion, the term “embryonic” refers to embryosderived by somatic cell nuclear transfer.

As used herein, “multipotent” or “multipotent cell” refers to a celltype that can give rise to a limited number of other particular celltypes.

As used herein, “expression” refers to the production of a material orsubstance as well as the level or amount of production of a material orsubstance. Thus, determining the expression of a specific marker refersto detecting either the relative or absolute amount of the marker thatis expressed or simply detecting the presence or absence of the marker.

As used herein, “marker” refers to any molecule that can be observed ordetected. For example, a marker can include, but is not limited to, anucleic acid, such as a transcript of a specific gene, a polypeptideproduct of a gene, a non-gene product polypeptide, a glycoprotein, acarbohydrate, a glycolipd, a lipid, a lipoprotein or a small molecule(for example, molecules having a molecular weight of less than 10,000amu)

When used in connection with cell cultures and/or cell populations, theterm “portion” means any non-zero amount of the cell culture or cellpopulation, which ranges from a single cell to the entirety of the cellculture or cells population.

With respect to cells in cell cultures or in cell populations, thephrase “substantially free of” means that the specified cell type ofwhich the cell culture or cell population is free, is present in anamount of less than about 5% of the total number of cells present in thecell culture or cell population.

With respect to cell culture medium, as used herein, “low serum RPMI”refers to a low serum containing medium, wherein the serum concentrationis gradually increased over a defined time period. For example, in oneembodiment, low serum RPMI comprises a concentration of about 0.2% fetalbovine serum (FBS) on the first day of cell growth, about 0.5% FBS onthe second day of cell growth and about 2% FBS on the third throughfifth day of cell growth. In another embodiment, low serum RPMIcomprises a concentration of about 0% on day one, about 0.2% on day twoand about 2% on the third and subsequent days.

As used herein, the terms “bFGF” and “FGF2” are used interchangeably.

Definitive Endoderm Cells and Processes Related Thereto

Embodiments described herein relate to novel, defined processes for theproduction of definitive endoderm cells in culture by differentiatingpluripotent cells, such as stem cells into multipotent definitiveendoderm cells. As described above, definitive endoderm cells do notdifferentiate into tissues produced from ectoderm or mesoderm, butrather, differentiate into the gut tube as well as organs that arederived from the gut tube. In certain preferred embodiments, thedefinitive endoderm cells are derived from hESCs. Such processes canprovide the basis for efficient production of human endodermal derivedtissues such as pancreas, liver, lung, stomach, intestine, thyroid andthymus. For example, production of definitive endoderm may be the firststep in differentiation of a stem cell to a functional insulin-producingβ-cell. To obtain useful quantities of insulin-producing β-cells, highefficiency of differentiation is desirable for each of thedifferentiation steps that occur prior to reaching the pancreaticislet/β-cell fate. Since differentiation of stem cells to definitiveendoderm cells represents perhaps the earliest step towards theproduction of functional pancreatic islet/β-cells (as shown in FIG. 1),high efficiency of differentiation at this step is particularlydesirable.

In view of the desirability of efficient differentiation of pluripotentcells to definitive endoderm cells, some aspects of the differentiationprocesses described herein relate to in vitro methodology that resultsin approximately 50-80% conversion of pluripotent cells to definitiveendoderm cells. Typically, such methods encompass the application ofculture and growth factor conditions in a defined and temporallyspecified fashion. Further enrichment of the cell population fordefinitive endoderm cells can be achieved by isolation and/orpurification of the definitive endoderm cells from other cells in thepopulation by using a reagent that specifically binds to definitiveendoderm cells. As such, some embodiments described herein relate todefinitive endoderm cells as well as methods for producing and isolatingand/or purifying such cells.

In order to determine the amount of definitive endoderm cells in a cellculture or cell population, a method of distinguishing this cell typefrom the other cells in the culture or in the population is desirable.Accordingly, certain embodiments described herein relate to cell markerswhose presence, absence and/or relative expression levels are specificfor definitive endoderm and methods for detecting and determining theexpression of such markers.

In some embodiments described herein, the presence, absence and/or levelof expression of a marker is determined by quantitative PCR (Q-PCR). Forexample, the amount of transcript produced by certain genetic markers,such as SOX17, CXCR4, OCT4, AFP, TM, SPARC, SOX7, MIXL1, GATA4, HNF3b,GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, CRIP1 and other markers describedherein is determined by quantitative Q-PCR. In other embodiments,immunohistochemistry is used to detect the proteins expressed by theabove-mentioned genes. In still other embodiments, Q-PCR andimmunohistochemical techniques are both used to identify and determinethe amount or relative proportions of such markers.

By using methods, such as those described above, to determine theexpression of one or more appropriate markers, it is possible toidentify definitive endoderm cells, as well as determine the proportionof definitive endoderm cells in a cell culture or cell population. Forexample, in some embodiments of the present invention, the definitiveendoderm cells or cell populations that are produced express the SOX17and/or the CXCR4 gene at a level of about 2 orders of magnitude greaterthan non-definitive endoderm cell types or cell populations. In otherembodiments, the definitive endoderm cells or cell populations that areproduced express the SOX17 and/or the CXCR4 gene at a level of more than2 orders of magnitude greater than non-definitive endoderm cell types orcell populations. In still other embodiments, the definitive endodermcells or cell populations that are produced express one or more of themarkers selected from the group consisting of SOX17, CXCR4, GSC, FGF17,VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 at a level of about 2 or more than 2orders of magnitude greater than non-definitive endoderm cell types orcell populations. In some embodiments described herein, definitiveendoderm cells do not substantially express PDX1.

Embodiments described herein also relate to definitive endodermcompositions. For example, some embodiments relate to cell culturescomprising definitive endoderm, whereas others relate to cellpopulations enriched in definitive endoderm cells. Some preferredembodiments relate to cell cultures which comprise definitive endodermcells, wherein at least about 50-80% of the cells in culture aredefinitive endoderm cells. An especially preferred embodiment relates tocells cultures comprising human cells, wherein at least about 50-80% ofthe human cells in culture are definitive endoderrn cells. Because theefficiency of the differentiation procedure can be adjusted by modifyingcertain parameters, which include but are not limited to, cell growthconditions, growth factor concentrations and the timing of culturesteps, the differentiation procedures described herein can result inabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orgreater than about 95% conversion of pluripotent cells to definitiveendoderm. In other preferred embodiments, conversion of a pluripotentcell population, such as a stem cell population, to substantially puredefinitive endoderm cell population is contemplated.

The compositions and methods described herein have several usefulfeatures. For example, the cell cultures and cell populations comprisingdefinitive endoderm as well as the methods for producing such cellcultures and cell populations are useful for modeling the early stagesof human development. Furthermore, the compositions and methodsdescribed herein can also serve for therapeutic intervention in diseasestates, such as diabetes mellitus. For example, since definitiveendoderm serves as the source for only a limited number of tissues, itcan be used in the development of pure tissue or cell types.

Production of Definitive Endoderm from Pluripotent Cells

Processes for differentiating pluripotent cells to produce cell culturesand enriched cell populations comprising definitive endoderm isdescribed below and in U.S. patent Ser. No. 11/021,618, entitledDEFINITIVE ENDODERM, filed Dec. 23, 2004, the disclosure of which isincorporated herein by reference in its entirety. In some of theseprocesses, the pluripotent cells used as starting material are stemcells. In certain processes, definitive endoderm cell cultures andenriched cell populations comprising definitive endoderm cells areproduced from embryonic stem cells. A preferred method for derivingdefinitive endoderm cells utilizes human embryonic stem cells as thestarting material for definitive endoderm production. Such pluripotentcells can be cells that originate from the morula, embryonic inner cellmass or those obtained from embryonic gonadal ridges. Human embryonicstem cells can be maintained in culture in a pluripotent state withoutsubstantial differentiation using methods that are known in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,453,357,5,670,372, 5,690,926 5,843,780, 6,200,806 and 6,251,671 the disclosuresof which are incorporated herein by reference in their entireties.

In some processes for producing definitive endoderm cells, hESCs aremaintained on a feeder layer. In such processes, any feeder layer whichallows hESCs to be maintained in a pluripotent state can be used. Onecommonly used feeder layer for the cultivation of human embryonic stemcells is a layer of mouse fibroblasts. More recently, human fibroblastfeeder layers have been developed for use in the cultivation of hESCs(see US Patent Application No. 2002/0072117, the disclosure of which isincorporated herein by reference in its entirety). Alternative processesfor producing definitive endoderm permit the maintenance of pluripotenthESC without the use of a feeder layer. Methods of maintainingpluripotent hESCs under feeder-free conditions have been described in USPatent Application No. 2003/0175956, the disclosure of which isincorporated herein by reference in its entirety.

The human embryonic stem cells used herein can be maintained in cultureeither with or without serum. In some embryonic stem cell maintenanceprocedures, serum replacement is used. In others, serum free culturetechniques, such as those described in US Patent Application No.2003/0190748, the disclosure of which is incorporated herein byreference in its entirety, are used.

Stem cells are maintained in culture in a pluripotent state by routinepassage until it is desired that they be differentiated into definitiveendoderm. In some processes, differentiation to definitive endoderm isachieved by providing to the stem cell culture a growth factor of theTGFβ superfamily in an amount sufficient to promote differentiation todefinitive endoderm. Growth factors of the TGFβ superfamily which areuseful for the production of definitive endoderm are selected from theNodal/Activin or BMP subgroups. In some preferred differentiationprocesses, the growth factor is selected from the group consisting ofNodal, activin A, activin B and BMP4. Additionally, the growth factorWnt3a and other Wnt family members are useful for the production ofdefinitive endoderm cells. In certain differentiation processes,combinations of any of the above-mentioned growth factors can be used.

With respect to some of the processes for the differentiation ofpluripotent stem cells to definitive endoderm cells, the above-mentionedgrowth factors are provided to the cells so that the growth factors arepresent in the cultures at concentrations sufficient to promotedifferentiation of at least a portion of the stem cells to definitiveendoderm cells. In some processes, the above-mentioned growth factorsare present in the cell culture at a concentration of at least about 5ng/ml, at least about 10 ng/ml, at least about 25 ng/ml, at least about50 ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at leastabout 200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, atleast about 500 ng/ml, at least about 1000 ng/ml, at least about 2000ng/ml, at least about 3000 ng/ml, at least about 4000 ng/ml, at leastabout 5000 ng/ml or more than about 5000 ng/ml.

In certain processes for the differentiation of pluripotent stem cellsto definitive endoderm cells, the above-mentioned growth factors areremoved from the cell culture subsequent to their addition. For example,the growth factors can be removed within about one day, about two days,about three days, about four days, about five days, about six days,about seven days, about eight days, about nine days or about ten daysafter their addition. In a preferred processes, the growth factors areremoved about four days after their addition.

Cultures of definitive endoderm cells can be grown in medium containingreduced serum or no serum. Under certain culture conditions, serumconcentrations can range from about 0.05% v/v to about 20% v/v. Forexample, in some differentiation processes, the serum concentration ofthe medium can be less than about 0.05% (v/v), less than about 0.1%(v/v), less than about 0.2% (v/v), less than about 0.3% (v/v), less thanabout 0.4% (v/v), less than about 0.5% (v/v), less than about 0.6%(v/v), less than about 0.7% (v/v), less than about 0.8% (v/v), less thanabout 0.9% (v/v), less than about 1% (v/v), less than about 2% (v/v),less than about 3% (v/v), less than about 4% (v/v), less than about 5%(v/v), less than about 6% (v/v), less than about 7% (v/v), less thanabout 8% (v/v), less than about 9% (v/v), less than about 10% (v/v),less than about 15% (v/v) or less than about 20% (v/v). In someprocesses, definitive endoderm cells are grown without serum or withserum replacement. In still other processes, definitive endoderm cellsare grown in the presence of B27. In such processes, the concentrationof B27 supplement can range from about 0.1% v/v to about 20% v/v.

Monitoring the Differentiation of Pluripotent Cells to DefinitiveEndoderm

The progression of the hESC culture to definitive endoderm can bemonitored by determining the expression of markers characteristic ofdefinitive endoderm. In some processes, the expression of certainmarkers is determined by detecting the presence or absence of themarker. Alternatively, the expression of certain markers can bedetermined by measuring the level at which the marker is present in thecells of the cell culture or cell population. In such processes, themeasurement of marker expression can be qualitative or quantitative. Onemethod of quantitating the expression of markers that are produced bymarker genes is through the use of quantitative PCR (Q-PCR). Methods ofperforming Q-PCR are well known in the art. Other methods which areknown in the art can also be used to quantitate marker gene expression.For example, the expression of a marker gene product can be detected byusing antibodies specific for the marker gene product of interest. Incertain processes, the expression of marker genes characteristic ofdefinitive endoderm as well as the lack of significant expression ofmarker genes characteristic of hESCs and other cell types is determined.

As described further in the Examples below, a reliable marker ofdefinitive endoderm is the SOX17 gene. As such, the definitive endodermcells produced by the processes described herein express the SOX17marker gene, thereby producing the SOX17 gene product. Other markers ofdefinitive endoderm are MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1. Since definitive endoderm cells express theSOX17 marker gene at a level higher than that of the SOX7 marker gene,which is characteristic of primitive and visceral endoderm (see Table1), in some processes, the expression of both SOX17 and SOX7 ismonitored. In other processes, expression of the both the SOX17 markergene and the OCT4 marker gene, which is characteristic of hESCs, ismonitored. Additionally, because definitive endoderm cells express theSOX17 marker gene at a level higher than that of the AFP, SPARC orThrombomodulin (TM) marker genes, the expression of these genes can alsobe monitored.

Another marker of definitive endoderm is the CXCR4 gene. The CXCR4 geneencodes a cell surface chemokine receptor whose ligand is thechemoattractant SDF-1. The principal roles of the CXCR4 receptor-bearingcells in the adult are believed to be the migration of hematopoeticcells to the bone marrow, lymphocyte trafficking and the differentiationof various B cell and macrophage blood cell lineages [Kim, C., andBroxmeyer, H. J. Leukocyte Biol. 65, 6-15 (1999)]. The CXCR4 receptoralso functions as a coreceptor for the entry of HIV-1 into T-cells[Feng, Y., et al. Science, 272, 872-877 (1996)]. In an extensive seriesof studies carried out by [McGrath, K. E. et al. Dev. Biology 213,442-456 (1999)], the expression of the chemokine receptor CXCR4 and itsunique ligand, SDF-1 [Kim, C., and Broxmyer, H., J. Leukocyte Biol. 65,6-15 (1999)], were delineated during early development and adult life inthe mouse. The CXCR4/SDF1 interaction in development became apparentwhen it was demonstrated that if either gene was disrupted in transgenicmice [Nagasawa et al. Nature, 382, 635-638 (1996)], Ma, Q., et alImmunity, 10, 463-471 (1999)] it resulted in late embryonic lethality.McGrath et al. demonstrated that CXCR4 is the most abundant chemokinereceptor messenger RNA detected during early gastrulating embryos (E7.5)using a combination of RNase protection and in situ hybridizationmethodologies. In the gastrulating embryo, CXCR4/SDF-1 signaling appearsto be mainly involved in inducing migration of primitive-streakgermlayer cells and is expressed on definitive endoderm, mesoderm andextraembryonic mesoderm present at this time. In E7.2-7.8 mouse embryos,CXCR4 and alpha-fetoprotein are mutually exclusive indicating a lack ofexpression in visceral endoderm [McGrath, K. E. et al. Dev. Biology 213,442-456 (1999)].

Since definitive endoderm cells produced by differentiating pluripotentcells express the CXCR4 marker gene, expression of CXCR4 can bemonitored in order to track the production of definitive endoderm cells.Additionally, definitive endoderm cells produced by the methodsdescribed herein express other markers of definitive endoderm including,but not limited to, SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1. Since definitive endoderm cells express theCXCR4 marker gene at a level higher than that of the SOX7 marker gene,the expression of both CXCR4 and SOX7 can be monitored. In otherprocesses, expression of both the CXCR4 marker gene and the OCT4 markergene, is monitored. Additionally, because definitive endoderm cellsexpress the CXCR4 marker gene at a level higher than that of the AFP,SPARC or Thrombomodulin (TM) marker genes, the expression of these genescan also be monitored.

It will be appreciated that expression of CXCR4 in endodermal cells doesnot preclude the expression of SOX17. As such, definitive endoderm cellsproduced by the processes described herein will substantially expressSOX17 and CXCR4 but will not substantially express AFP, TM, SPARC orPDX1.

It will be appreciated that SOX17 and/or CXCR4 marker expression isinduced over a range of different levels in definitive endoderm cellsdepending on the differentiation conditions. As such, in someembodiments described herein, the expression of the SOX17 marker and/orthe CXCR4 marker in definitive endoderm cells or cell populations is atleast about 2-fold higher to at least about 10,000-fold higher than theexpression of the SOX17 marker and/or the CXCR4 marker in non-definitiveendoderm cells or cell populations, for example pluripotent stem cells.In other embodiments, the expression of the SOX17 marker and/or theCXCR4 marker in definitive endoderm cells or cell populations is atleast about 4-fold higher, at least about 6-fold higher, at least about8-fold higher, at least about 10-fold higher, at least about 15-foldhigher, at least about 20-fold higher, at least about 40-fold higher, atleast about 80-fold higher, at least about 100-fold higher, at leastabout 150-fold higher, at least about 200-fold higher, at least about500-fold higher, at least about 750-fold higher, at least about1000-fold higher, at least about 2500-fold higher, at least about5000-fold higher, at least about 7500-fold higher or at least about10,000-fold higher than the expression of the SOX17 marker and/or theCXCR4 marker in non-definitive endoderm cells or cell populations, forexample pluripotent stem cells. In some embodiments, the expression ofthe SOX17 marker and/or CXCR4 marker in definitive endoderm cells orcell populations is infinitely higher than the expression of the SOX17marker and/or the CXCR4 marker in non-definitive endoderm cells or cellpopulations, for example pluripotent stem cells.

It will also be appreciated that in some embodiments described herein,the expression of markers selected from the group consisting of GATA4,MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 indefinitive endoderm cells or cell populations is increased as comparedto the expression of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1,CMKOR1 and CRIP1 in non-definitive endoderm cells or cell populations.

Additionally, it will be appreciated that there is a range ofdifferences between the expression level of the SOX17 marker and theexpression levels of the OCT4, SPARC, AFP, TM and/or SOX7 markers indefinitive endoderm cells. Similarly, there exists a range ofdifferences between the expression level of the CXCR4 marker and theexpression levels of the OCT4, SPARC, AFP, TM and/or SOX7 markers indefinitive endoderm cells. As such, in some embodiments describedherein, the expression of the SOX17 marker or the CXCR4 marker is atleast about 2-fold higher to at least about 10,000-fold higher than theexpression of OCT4, SPARC, AFP, TM and/or SOX7 markers. In otherembodiments, the expression of the SOX17 marker or the CXCR4 marker isat least about 4-fold higher, at least about 6-fold higher, at leastabout 8-fold higher, at least about 10-fold higher, at least about15-fold higher, at least about 20-fold higher, at least about 40-foldhigher, at least about 80-fold higher, at least about 100-fold higher,at least about 150-fold higher, at least about 200-fold higher, at leastabout 500-fold higher, at least about 750-fold higher, at least about1000-fold higher, at least about 2500-fold higher, at least about5000-fold higher, at least about 7500-fold higher or at least about10,000-fold higher than the expression of OCT4, SPARC, AFP, TM and/orSOX7 markers. In some embodiments, OCT4, SPARC, AFP, TM and/or SOX7markers are not significantly expressed in definitive endoderm cells.

It will also be appreciated that in some embodiments described herein,the expression of markers selected from the group consisting of GATA4,MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 indefinitive endoderm cells is increased as compared to the expression ofOCT4, SPARC, AFP, TM and/or SOX7 in definitive endoderm cells.

Enrichment, Isolation and/or Purification of Definitive Endoderm

Definitive endoderm cells produced by any of the above-describedprocesses can be enriched, isolated and/or purified by using an affinitytag that is specific for such cells. Examples of affinity tags specificfor definitive endoderm cells are antibodies, ligands or other bindingagents that are specific to a marker molecule, such as a polypeptide,that is present on the cell surface of definitive endoderm cells butwhich is not substantially present on other cell types that would befound in a cell culture produced by the methods described herein. Insome processes, an antibody which binds to CXCR4 is used as an affinitytag for the enrichment, isolation or purification of definitive endodermcells. In other processes, the chemokine SDF-1 or other molecules basedon SDF-1 can also be used as affinity tags. Such molecules include, butnot limited to, SDF-1 fragments, SDF-1 fusions or SDF-1 mimetics.

Methods for making antibodies and using them for cell isolation areknown in the art and such methods can be implemented for use with theantibodies and definitive endoderm cells described herein. In oneprocess, an antibody which binds to CXCR4 is attached to a magnetic beadand then allowed to bind to definitive endoderm cells in a cell culturewhich has been enzymatically treated to reduce intercellular andsubstrate adhesion. The cell/antibody/bead complexes are then exposed toa movable magnetic field which is used to separate bead-bound definitiveendoderm cells from unbound cells. Once the definitive endoderm cellsare physically separated from other cells in culture, the antibodybinding is disrupted and the cells are replated in appropriate tissueculture medium.

Additional methods for obtaining enriched, isolated or purifieddefinitive endoderm cell cultures or populations can also be used. Forexample, in some embodiments, the CXCR4 antibody is incubated with adefinitive endoderm-containing cell culture that has been treated toreduce intercellular and substrate adhesion. The cells are then washed,centrifuged and resuspended. The cell suspension is then incubated witha secondary antibody, such as an FITC-conjugated antibody that iscapable of binding to the primary antibody. The cells are then washed,centrifuged and resuspended in buffer. The cell suspension is thenanalyzed and sorted using a fluorescence activated cell sorter (FACS).CXCR4-positive cells are collected separately from CXCR4-negative cells,thereby resulting in the isolation of such cell types. If desired, theisolated cell compositions can be further purified by using an alternateaffinity-based method or by additional rounds of sorting using the sameor different markers that are specific for definitive endoderm.

In still other processes, definitive endoderm cells are enriched,isolated and/or purified using a ligand or other molecule that binds toCXCR4. In some processes, the molecule is SDF-1 or a fragment fusion ormimetic thereof.

In some embodiments of the processes described herein, definitiveendoderm cells are fluorescently labeled then isolated from non-labeledcells by using a fluorescence activated cell sorter (FACS). In suchembodiments, a nucleic acid encoding green fluorescent protein (GFP) oranother nucleic acid encoding an expressible fluorescent marker gene isused to label PDX1-positive cells. For example, in some embodiments, atleast one copy of a nucleic acid encoding GFP or a biologically activefragment thereof is introduced into a pluripotent cell, preferably ahuman embryonic stem cell, downstream of the SOX17 or CXCR4 promotersuch that the expression of the GFP gene product or biologically activefragment thereof is under control of the SOX17 or CXCR4 promoter. Insome embodiments, the entire coding region of the nucleic acid, whichencodes SOX17 or CXCR4, is replaced by a nucleic acid encoding GFP or abiologically active fragment thereof. In other embodiments, the nucleicacid encoding CFP or a biologically active fragment thereof is fused inframe with at least a portion of the nucleic acid encoding SOX17 orCXCR4, thereby generating a fusion protein. In such embodiments, thefusion protein retains a fluorescent activity similar to GFP.

Fluorescently marked cells, such as the above-described pluripotentcells, are differentiated to definitive endoderm as described previouslyabove. Because definitive endoderm cells express the fluorescent markergene, whereas other cell types do not, definitive endoderm cells can beseparated from the other cell types. In some embodiments, cellsuspensions comprising a mixture of fluorescently-labeled definitiveendoderm cells and unlabeled non-definitive endoderm cells are sortedusing a FACS. Definitive endoderm cells are collected separately fromnon-fluorescing cells, thereby resulting in the isolation of definitiveendoderm. If desired, the isolated cell compositions can be furtherpurified by additional rounds of sorting using the same or differentmarkers that are specific for definitive endoderm.

In preferred processes, definitive endoderm cells are enriched, isolatedand/or purified from other non-definitive endoderm cells after the stemcell cultures are induced to differentiate towards the definitiveendoderm lineage. It will be appreciated that the above-describedenrichment, isolation and purification procedures can be used with suchcultures at any stage of differentiation.

In addition to the procedures just described, definitive endoderm cellsmay also be isolated by other techniques for cell isolation.Additionally, definitive endoderm cells may also be enriched or isolatedby methods of serial subculture in growth conditions which promote theselective survival or selective expansion of the definitive endodermcells.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of definitive endoderm cells and or tissues can be producedin vitro from pluripotent cell cultures or cell populations, such asstem cell cultures or populations, which have undergone at least somedifferentiation. In some methods, the cells undergo randomdifferentiation. In a preferred method, however, the cells are directedto differentiate primarily into definitive endoderm. Some preferredenrichment, isolation and/or purification methods relate to the in vitroproduction of definitive endoderm from human embryonic stem cells.

Using the methods described herein, cell populations or cell culturescan be enriched in definitive endoderm content by at least about 2- toabout 1000-fold as compared to untreated cell populations or cellcultures. In some embodiments, definitive endoderm cells can be enrichedby at least about 5- to about 500-fold as compared to untreated cellpopulations or cell cultures. In other embodiments, definitive endodermcells can be enriched from at least about 10- to about 200-fold ascompared to untreated cell populations or cell cultures. In still otherembodiments, definitive endoderm cells can be enriched from at leastabout 20- to about 100-fold as compared to untreated cell populations orcell cultures. In yet other embodiments, definitive endoderm cells canbe enriched from at least about 40- to about 80-fold as compared tountreated cell populations or cell cultures. In certain embodiments,definitive endoderm cells can be enriched from at least about 2- toabout 20-fold as compared to untreated cell populations or cellcultures.

Compositions Comprising Definitive Endoderm

Cell compositions produced by the above-described methods include cellcultures comprising definitive endoderm and cell populations enriched indefinitive endoderm. For example, cell cultures which comprisedefinitive endoderm cells, wherein at least about 50-80% of the cells inculture are definitive endoderm cells, can be produced. Because theefficiency of the differentiation process can be adjusted by modifyingcertain parameters, which include but are not limited to, cell growthconditions, growth factor concentrations and the timing of culturesteps, the differentiation procedures described herein can result inabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orgreater than about 95% conversion of pluripotent cells to definitiveendoderm. In processes in which isolation of definitive endoderm cellsis employed, for example, by using an affinity reagent that binds to theCXCR4 receptor, a substantially pure definitive endoderm cell populationcan be recovered.

Some embodiments described herein relate to compositions, such as cellpopulations and cell cultures, that comprise both pluripotent cells,such as stem cells, and definitive endoderm cells. For example, usingthe methods described herein, compositions comprising mixtures of hESCsand definitive endoderm cells can be produced. In some embodiments,compositions comprising at least about 5 definitive endoderm cells forabout every 95 pluripotent cells are produced. In other embodiments,compositions comprising at least about 95 definitive endoderm cells forabout every 5 pluripotent cells are produced. Additionally, compositionscomprising other ratios of definitive endoderm cells to pluripotentcells are contemplated. For example, compositions comprising at leastabout 1 definitive endoderm cell for about every 1,000,000 pluripotentcells, at least about 1 definitive endoderm cell for about every 100,000pluripotent cells, at least about 1 definitive endoderm cell for aboutevery 10,000 pluripotent cells, at least about 1 definitive endodermcell for about every 1000 pluripotent cells, at least about 1 definitiveendoderm cell for about every 500 pluripotent cells, at least about 1definitive endoderm cell for about every 100 pluripotent cells, at leastabout 1 definitive endoderm cell for about every 10 pluripotent cells,at least about 1 definitive endoderm cell for about every 5 pluripotentcells, at least about 1 definitive endoderm cell for about every 2pluripotent cells, at least about 2 definitive endoderm cells for aboutevery 1 pluripotent cell, at least about 5 definitive endoderm cells forabout every 1 pluripotent cell, at least about 10 definitive endodermcells for about every 1 pluripotent cell, at least about 20 definitiveendoderm cells for about every 1 pluripotent cell, at least about 50definitive endoderm cells for about every 1 pluripotent cell, at leastabout 100 definitive endoderm cells for about every 1 pluripotent cell,at least about 1000 definitive endoderm cells for about every 1pluripotent cell, at least about 10,000 definitive endoderm cells forabout every 1 pluripotent cell, at least about 100,000 definitiveendoderm cells for about every 1 pluripotent cell and at least about1,000,000 definitive endoderm cells for about every 1 pluripotent cellare contemplated. In some embodiments, the pluripotent cells are humanpluripotent stem cells. In certain embodiments the stem cells arederived from a morula, the inner cell mass of an embryo or the gonadalridges of an embryo. In certain other embodiments, the pluripotent cellsare derived from the gondal or germ tissues of a multicellular structurethat has developed past the embryonic stage.

Some embodiments described herein relate to cell cultures or cellpopulations comprising from at least about 5% definitive endoderm cellsto at least about 95% definitive endoderm cells. In some embodiments thecell cultures or cell populations comprise mammalian cells. In preferredembodiments, the cell cultures or cell populations comprise human cells.For example, certain specific embodiments relate to cell culturescomprising human cells, wherein from at least about 5% to at least about95% of the human cells are definitive endoderm cells. Other embodimentsrelate to cell cultures comprising human cells, wherein at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90% or greaterthan 90% of the human cells are definitive endoderm cells. Inembodiments where the cell cultures or cell populations comprise humanfeeder cells, the above percentages are calculated without respect tothe human feeder cells in the cell cultures or cell populations.

Further embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising human cells, such as humandefinitive endoderm cells, wherein the expression of either the SOX17 orthe CXCR4 marker is greater than the expression of the OCT4, SPARC,alpha-fetoprotein (AFP), Thrombomodulin (TM) and/or SOX7 marker in atleast about 5% of the human cells. In other embodiments, the expressionof either the SOX17 or the CXCR4 marker is greater than the expressionof the OCT4, SPARC, AFP, TM and/or SOX7 marker in at least about 10% ofthe human cells, in at least about 15% of the human cells, in at leastabout 20% of the human cells, in at least about 25% of the human cells,in at least about 30% of the human cells, in at least about 35% of thehuman cells, in at least about 40% of the human cells, in at least about45% of the human cells, in at least about 50% of the human cells, in atleast about 55% of the human cells, in at least about 60% of the humancells, in at least about 65% of the human cells, in at least about 70%of the human cells, in at least about 75% of the human cells, in atleast about 80% of the human cells, in at least about 85% of the humancells, in at least about 90% of the human cells, in at least about 95%of the human cells or in greater than 95% of the human cells. Inembodiments where the cell cultures or cell populations comprise humanfeeder cells, the above percentages are calculated without respect tothe human feeder cells in the cell cultures or cell populations.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations, comprisinghuman cells, such as human definitive endoderm cells, wherein theexpression of one or more markers selected from the group consisting ofGATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 isgreater than the expression of the OCT4, SPARC, AFP, TM and/or SOX7markers in from at least about 5% to greater than at least about 95% ofthe human cells. In embodiments where the cell cultures or cellpopulations comprise human feeder cells, the above percentages arecalculated without respect to the human feeder cells in the cellcultures or cell populations.

Still other embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising human cells, such as humandefinitive endoderm cells, wherein the expression both the SOX17 and theCXCR4 marker is greater than the expression of the OCT4, SPARC, AFP, TMand/or SOX7 marker in at least about 5% of the human cells. In otherembodiments, the expression of both the SOX17 and the CXCR4 marker isgreater than the expression of the OCT4, SPARC, AFP, TM and/or SOX7marker in at least about 10% of the human cells, in at least about 15%of the human cells, in at least about 20% of the human cells, in atleast about 25% of the human cells, in at least about 30% of the humancells, in at least about 35% of the human cells, in at least about 40%of the human cells, in at least about 45% of the human cells, in atleast about 50% of the human cells, in at least about 55% of the humancells, in at least about 60% of the human cells, in at least about 65%of the human cells, in at least about 70% of the human cells, in atleast about 75% of the human cells, in at least about 80% of the humancells, in at least about 85% of the human cells, in at least about 90%of the human cells, in at least about 95% of the human cells or ingreater than 95% of the human cells. In embodiments where the cellcultures or cell populations comprise human feeder cells, the abovepercentages are calculated without respect to the human feeder cells inthe cell cultures or cell populations.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations, comprisinghuman cells, such as human definitive endoderm cells, wherein theexpression of the GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1,CMKOR1 and CRIP1 markers is greater than the expression of the OCT4,SPARC, AFP, TM and/or SOX7 markers in from at least about 5% to greaterthan at least about 95% of the human cells. In embodiments where thecell cultures or cell populations comprise human feeder cells, the abovepercentages are calculated without respect to the human feeder cells inthe cell cultures or cell populations.

Additional embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising mammalian endodermalcells, such as human endoderm cells, wherein the expression of eitherthe SOX17 or the CXCR4 marker is greater than the expression of theOCT4, SPARC, AFP, TM and/or SOX7 marker in at least about 5% of theendodermal cells. In other embodiments, the expression of either theSOX17 or the CXCR4 marker is greater than the expression of the OCT4,SPARC, AFP, TM and/or SOX7 marker in at least about 10% of theendodermal cells, in at least about 15% of the endodermal cells, in atleast about 20% of the endodermal cells, in at least about 25% of theendodermal cells, in at least about 30% of the endodermal cells, in atleast about 35% of the endodermal cells, in at least about 40% of theendodermal cells, in at least about 45% of the endodermal cells, in atleast about 50% of the endodermal cells, in at least about 55% of theendodermal cells, in at least about 60% of the endodermal cells, in atleast about 65% of the endodermal cells, in at least about 70% of theendodermal cells, in at least about 75% of the endodermal cells, in atleast about 80% of the endodermal cells, in at least about 85% of theendodermal cells, in at least about 90% of the endodermal cells, in atleast about 95% of the endodermal cells or in greater than 95% of theendodermal cells.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations comprisingmammalian endodermal cells, wherein the expression of one or moremarkers selected from the group consisting of GATA4, MIXL1, HNF3b, GSC,FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is greater than theexpression of the OCT4, SPARC, AFP, TM and/or SOX7 markers in from atleast about 5% to greater than at least about 95% of the endodermalcells.

Still other embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising mammalian endodermalcells, such as human endodermal cells, wherein the expression of boththe SOX17 and the CXCR4 marker is greater than the expression of theOCT4, SPARC, AFP, TM and/or SOX7 marker in at least about 5% of theendodermal cells. In other embodiments, the expression of both the SOX17and the CXCR4 marker is greater than the expression of the OCT4, SPARC,AFP, TM and/or SOX7 marker in at least about 10% of the endodermalcells, in at least about 15% of the endodermal cells, in at least about20% of the endodermal cells, in at least about 25% of the endodermalcells, in at least about 30% of the endodermal cells, in at least about35% of the endodermal cells, in at least about 40% of the endodermalcells, in at least about 45% of the endodermal cells, in at least about50% of the endodermal cells, in at least about 55% of the endodermalcells, in at least about 60% of the endodermal cells, in at least about65% of the endodermal cells, in at least about 70% of the endodermalcells, in at least about 75% of the endodermal cells, in at least about80% of the endodermal cells, in at least about 85% of the endodermalcells, in at least about 90% of the endodermal cells, in at least about95% of the endodermal cells or in greater than 95% of the endodermalcells.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations comprisingmammalian endodermal cells, wherein the expression of the GATA4, MIXL1,HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 markers isgreater than the expression of the OCT4, SPARC, AFP, TM and/or SOX7markers in from at least about 5% to greater than at least about 95% ofthe endodermal cells.

Using the methods described herein, compositions comprising definitiveendoderm cells substantially free of other cell types can be produced.In some embodiments described herein, the definitive endoderm cellpopulations or cell cultures produced by the methods described hereinare substantially free of cells that significantly express the OCT4,SOX7, AFP, SPARC, TM, ZIC1 or BRACH marker genes.

In one embodiment, a description of a definitive endoderm cell based onthe expression of marker genes is, SOX17 high, MIXL1 high, AFP low,SPARC low, Thrombomodulin low, SOX7 low, CXCR4 high.

Expansion of Definitive Endoderm Cells

According to some of the in vitro methods described herein, definitiveendoderm cells are maintained, grown, passaged and/or expanded while incell culture. In some embodiments the definitive endoderm cells aremaintained, grown, passaged and/or expanded without any significantdifferentiation. In other words, in such embodiments, the definitiveendoderm cells maintain the definitive endoderm phenotype while beingmaintained, grown, passaged and/or expanded in cell culture.

In some embodiments, definitive endoderm cells used in the expansionmethods described herein, are multipotent cells that can differentiateinto cells of the gut tube or organs derived therefrom. Such cellsinclude, but are not limited to, cells of the pancreas, liver, lungs,stomach, intestine, thyroid, thymus, pharynx, gallbladder and urinarybladder as well as precursors of such cells. Additionally, these cellscan further develop into higher order structures such as tissues and/ororgans. In some embodiments, the definitive endoderm cells are humandefinitive endoderm cells.

Some embodiments of the methods described herein comprise a step ofobtaining a cell culture comprising definitive endoderm cells. The cellculture can be a pure culture of definitive endoderm cells or a mixedcell culture that comprises definitive endoderm cells as well as cellsof other types. For example, the cell culture can be a culturecomprising both definitive endoderm cells and human embryonic stem cells(hESCs). In some embodiments, the definitive endoderm cell culture isobtained by differentiating in vitro cell cultures of hESCs. In certainembodiments, the hESCs are derived from a morula, the inner cell mass ofan embryo or the gonadal ridges of an embryo. In certain otherembodiments, the pluripotent cells are derived from the gonadal or germtissues of a multicellular structure that has developed past theembryonic stage.

Methods of differentiating hESCs so as to produce cell culturescomprising human definitive endoderm cells have been describedthroughout this application and in U.S. patent application Ser. No.11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, thedisclosure of which is incorporated herein by reference in its entirety.However, it will be appreciated that any known methods for producinghuman definitive endoderm cells from hESCs or from other human celltypes can be used. In some embodiments described herein, the cultures ofdefinitive endoderm cells produced by differentiating hESCs can be mixeddefinitive endoderm cultures, which comprise definitive endoderm cellsand one or more types of other cells, enriched definitive endoderm cellcultures and/or purified definitive endoderm cell cultures. Some methodsfor obtaining definitive endoderm cells from hESCs comprise contactingor otherwise providing the hESCs with at least one growth factor fromthe TGFβ superfamily. Such growth factors can include, but are notlimited to, Nodal, activin A and activin B. In some embodiments, thegrowth factor is provided to the hESCs at a concentration ranging from 5ng/ml to 5000 ng/ml. In certain embodiments the growth factor isprovided to the hESCs in culture at a concentration of at least about 5ng/ml, at least about 10 ng/ml, at least about 25 ng/ml, at least about50 ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at leastabout 200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, atleast about 500 ng/ml, at least about 1000 ng/ml, at least about 2000ng/ml, at least about 3000 ng/ml at least about 4000 ng/ml, at leastabout 5000 ng/ml or more than about 5000 ng/ml.

In other embodiments of the methods described herein, definitiveendoderm cells can be obtained from a pre-existing culture of definitiveendoderm cells. In such embodiments, either a portion of or the entireculture may be used in the definitive endoderm expansion methodsdescribed herein.

In addition to obtaining cell cultures comprising definitive endodermcells, some embodiments of the expansion methods described herein alsocomprise the step of isolating at least some of the definitive endodermcells from the cell culture. In such embodiments, at least some of thedefinitive endoderm cells are separated from at least some of the othercells in the cell culture, thereby producing a cell population enrichedin definitive endoderm cells. In some embodiments, at least some of thedefinitive endoderm cell are removed from the cell culture while atleast some of the other cells remain in the cell culture. Other cellsthat are present in the cell culture can include, but are not limitedto, hESCs, primitive endoderm, trophectoderm, mesoderm and ectoderm.

In other embodiments described herein, the isolating step comprisesproviding the cells in the cell culture with a reagent which binds to amarker expressed in said definitive endoderm cells but which is notsubstantially expressed in said other cells present in the cell culture.As described previously herein, in some embodiments, the marker can beany cell surface marker that is specific to definitive endoderm cells.One such marker that is described throughout this application (seeespecially the Examples below) is the CXCR4 marker. As describedpreviously herein, the reagent-bound definitive endoderm cells can beseparated from the non-reagent-bound cells by numerous methods. Forexample, an antibody against the CXCR4 receptor that is selectivelypresent on the surface of definitive endoderm cells, can be provided todefinitive endoderm cells in a cell culture. Antibody-bound definitiveendoderm cells can then be separated from other cells in the culture by,for example, fluorescent activated cell sorting (FACS), binding theantibody to a solid support or isolating appropriately tagged antibodyin a magnetic field. In some embodiments, the antibody is released fromthe definitive endoderm cells after the separation process.

As an alternative means of separation, at least some of the definitiveendoderm cells are separated from at least some of the other cells inthe culture by specifically fluorescently labeling the definitiveendoderm cells in culture and then separating the labeled cells from theunlabeled cells by FACS. As described previously above and in theExamples, in such embodiments, hESCs are transfected with a vectorcomprising a fluorescent reporter gene under the control of the promoterof a marker gene that is highly expressed in definitive endoderm cellsbut not significantly expressed in other cell types. In someembodiments, the fluorescent reporter gene is the gene encoding greenfluorescent protein (GFP) or enhanced green fluorescent protein (EGFP).In some embodiments, the GFP and/or EGFP is expressed under the controlof the SOX17 or the CXCR4 promoter. Transfected hESCs are then grown inculture in the presence of a differentiation factor that specificallyinduces the production of definitive endoderm. In preferred embodiments,the differentiation factor is activin A. In other preferred embodiments,activin A is added to the cell culture at a concentration of 100 ng/ml.

In some embodiments described herein, the enriched definitive endodermcell populations that are produced as a result of the isolating step aresubstantially free of cells other than definitive endoderm cells. Inother embodiments, the enriched definitive endoderm cell populationscomprise from at least about 96% to at least about 100% definitiveendoderm cells. In still other embodiments, the enriched definitiveendoderm cell populations comprise from at least about 96%, at leastabout 97%, at least about 98%, at least about 99% and at least about100% definitive endoderm cells.

According to further embodiments of the expansion methods describedherein a cell culture step is contemplated. For example, someembodiments include a culturing step that comprises plating thepopulation enriched in definitive endoderm cells or a portion of thepopulation. In some embodiments, the cells are plated on a surfacecoated with human fibronectin. In other embodiments the plates arecoated with poly-ornithine. In still other embodiments, the plates arecoated with poly-ornithine and human fibronectin. In preferredembodiments, the plates are IVF plates coated with both poly-ornithineand human fibronectin. It will be appreciated that although humanfibronectin is a preferred coating for the plates described herein,fibronectin from other sources is sufficient for coating plates.

In other embodiments, the culturing step comprises incubating theenriched definitive endoderm cell population or portion thereof in anexpansion medium comprising about 2% (v/v) serum. In some embodiments,the serum concentration can range from about 0% (v/v) to about 20%(v/v). For example, in some methods described herein, the serumconcentration of the medium can be about 0.05% (v/v), about 0.1% (v/v),about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v),about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v),about 1% (v/v), about 2% (vfv), about 3% (v/v), about 4% (v/v), about 5%(v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v), about 9% (v/v),about 10% (v/v), about 15% (v/v) or about 20% (v/v). In someembodiments, serum replacement is included in the medium.

In still other embodiments of the expansion methods described herein,the expansion medium also comprises at least one growth factor. Incertain embodiments, the at least one growth factor is a growth factorcomprises a member of the TGFβ superfamily. In such embodiments, the atleast one growth factor of the TGFβ superfamily includes, but is notlimited to Nodal, activin A, activin B and combinations of these growthfactors. Alternatively, in some embodiments, the at least one growthfactor can be IGF1 or a combination of IGF and a growth factor of theTGFβ superfamily. In other embodiments, the at least one growth factorcan be bFGF, EGF or another growth factor. In yet other embodiments, theat least one growth factor can be a combination of bFGF, EGF and agrowth factor of the TGFβ superfamily. In each of the above embodiments,one or more of the growth factors can be present at a concentrationranging from about 1 ng/ml to about 5000 ng/ml. In such embodiments, theconcentration of growth factor in the medium is at least about 5 ng/ml,at least about 10 ng/ml, at least about 25 ng/ml, at least about 50ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at least about200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at leastabout 500 ng/ml, at least about 1000 ng/ml, at least about 2000 ng/ml,at least about 3000 ng/ml, at least about 4000 ng/ml, at least about5000 ng/ml or more than about 5000 ng/ml. In certain embodiments, acombination of growth factors is present in the culture medium. In suchembodiments, each growth factor is present in the medium at aconcentration of at a concentration of at least about 5 ng/ml, at leastabout 10 ng/ml, at least about 25 ng/ml, at least about 50 ng/ml, atleast about 75 ng/ml, at least about 100 ng/ml, at least about 200ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at leastabout 500 ng/ml, at least about 1000 ng/ml, at least about 2000 ng/ml,at least about 3000 ng/ml, at least about 4000 ng/ml, at least about5000 ng/ml or more than about 5000 ng/ml.

In addition to the above-described expansion methods, in someembodiments definitive endoderm cells are expanded by first obtaining acell culture comprising definitive endoderm cells and then passaging thedefinitive endoderm cells so as to produce a plurality of cell culturescomprising definitive endoderm cells. These methods of expandingdefinitive endoderm cells by passaging the cells can be performed usingany definitive endoderm culture regardless of how such culture isobtained. For example, these methods can be performed as part of theculturing step that follows the cell isolation step in theabove-described expansion methods, or alternatively, this methods can beperformed using definitive endoderm cells that have been freshlydifferentiated from hESCs.

In accordance with certain aspects of the expansion methods describedherein, the step of passaging definitive endoderm cells comprisesproviding at least one enzyme to a cell culture comprising definitiveendoderm cells. For example, the at least one enzyme can be one or moreenzymes selected from the group consisting of papain, pronase, type Icollagenase, type II collagenase, type III collagenase, type IVcollagenase, trypsin, hyaluronidase, elastase, DNase I, and dispase. Insome embodiments, the at least one enzyme comprises at least oneprotease. In preferred embodiments, the at least one protease comprisestrypsin. For example, in certain embodiments, definitive endoderm cellsgrowing in a culture vessel are passaged with trypsin by first removingthe culture medium from the cells. Next, a sterile trypsin solution isprovided to the definitive endoderm cells for several minutes at roomtemperature. The trypsin solution is then gently removed so as not todisturb the cells. After the trypsin solution has been removed, thedefinitive endoderm cells are provided with a culture medium, such asRPMI with 2% (v/v) serum, and the cell culture vessel is then agitatedso as to disrupt cell adhesions and generate a cell suspension. In someembodiments, the cell culture medium comprises trypsin inhibitor toinactivate residual trypsin.

It will be appreciated that trypsin can be provided in a variety ofsterile solutions, for example, trypsin can be provided to thedefinitive endoderm cells in a balanced salt solution, such as Hanksbalanced salt solution. Alternatively, trypsin can be provided to thedefinitive endoderm in a medium with or without serum, for example inlow serum RPMI.

In accordance with other aspects of the expansion methods describedherein, the step of passaging definitive endoderm cells comprisesmechanically disrupting contacts between said definitive endoderm cells.Such mechanical disruption techniques should be sufficient tosubstantially disrupt cell contacts and the substrate, however, thesetechniques should not be so harsh as to affect cell viability.Mechanical cell disruption techniques, such as trituration, are known tothose of ordinary skill in the art.

In accordance with yet other aspects of the expansion methods describedherein, the step of passaging definitive endoderm cells comprisesincubating said definitive endoderm cells in a cell dispersal buffer.The cell dispersal buffer can be any dispersal buffer known in the art,for example, commercially available chemical dissociation buffers.

In some embodiments of the expansion methods described herein, thedefinitive endoderm cells are grown in a cell culture vessel. Cellculture vessels can include, but are not limited to, tissue cultureflasks and cell culture plate, such as microtiter plates. In someembodiments, the definitive endoderm cells in culture are attached to asubstrate. In certain embodiments, the step of passaging said definitiveendoderm cells comprises detaching said definitive endoderm cells fromthe substrate. In preferred embodiments, the substrate is a surface of atissue culture flask. In other preferred embodiments, the substrate is asurface of a microtiter plate.

EXAMPLES

Many of the examples below describe the use of pluripotent human cells.Methods of producing pluripotent human cells are well known in the artand have been described numerous scientific publications, including U.S.Pat. Nos. 5,453,357, 5,670,372, 5,690,926, 6,090,622, 6,200,806 and6,251,671 as well as U.S. Patent Application Publication No.2004/0229350, the disclosures of which are incorporated herein byreference in their entireties.

Example 1 Human ES Cells

For our studies of endoderm development we employed human embryonic stemcells, which are pluripotent and can divide seemingly indefinitely inculture while maintaining a normal karyotype. ES cells were derived fromthe 5-day-old embryo inner cell mass using either immunological ormechanical methods for isolation. In particular, the human embryonicstem cell line hESCyt-25 was derived from a supernumerary frozen embryofrom an in vitro fertilization cycle following informed consent by thepatient. Upon thawing the hatched blastocyst was plated on mouseembryonic fibroblasts (MEF), in ES medium (DMEM, 20% FBS, non essentialamino acids, beta-mercaptoethanol, and FGF2). The embryo adhered to theculture dish and after approximately two weeks, regions ofundifferentiated hESCs were transferred to new dishes with MEFs.Transfer was accomplished with mechanical cutting and a brief digestionwith dispase, followed by mechanical removal of the cell clusters,washing and re-plating. Since derivation, hESCyt-25 has been seriallypassaged over 100 times. We employed the hESCyt-25 human embryonic stemcell line as our starting material for the production of definitiveendoderm.

It will be appreciated by those of skill in the art that stem cells orother pluripotent cells can also be used as starting material for thedifferentiation procedures described herein. For example, cells obtainedfrom embryonic gonadal ridges, which can be isolated by methods known inthe art, can be used as pluripotent cellular starting material.

Example 2 hESCyt-25 Characterization

The human embryonic stem cell line, hESCyt-25 has maintained a normalmorphology, karyotype, growth and self-renewal properties over 18 monthsin culture. This cell line displays strong immunoreactivity for theOCT4, SSEA-4 and TRA-1-60 antigens, all of which, are characteristic ofundifferentiated hESCs and displays alkaline phosphatase activity aswell as a morphology identical to other established hESC lines.Furthermore, the human stem cell line, hESCyt-25, also readily formsembryoid bodies (EBs) when cultured in suspension. As a demonstration ofits pluripotent nature, hESCyT-25 differentiates into various cell typesthat represent the three principal germ layers. Ectoderm production wasdemonstrated by Q-PCR for ZIC1 as well as immunocytochemistry (ICC) fornestin and more mature neuronal markers. Immunocytochemical staining forβ-III tubulin was observed in clusters of elongated cells,characteristic of early neurons. Previously, we treated EBs insuspension with retinoic acid, to induce differentiation of pluripotentstem cells to visceral endoderm (VE), an extra-embryonic lineage.Treated cells expressed high levels of α-fetoprotein (AFP) and. SOX7,two markers of VE, by 54 hours of treatment. Cells differentiated inmonolayer expressed AFP in sporadic patches as demonstrated byimmunocytochemical staining. As will be described below, the hESCyT-25cell line was also capable of forming definitive endoderm, as validatedby real-time quantitative polymerase chain reaction (Q-PCR) andimmunocytochemistry for SOX17, in the absence of AFP expression. Todemonstrate differentiation to mesoderm, differentiating EBs wereanalyzed for Brachyury gene expression at several time points. Brachyuryexpression increased progressively over the course of the experiment. Inview of the foregoing, the hESCyT-25 line is pluripotent as shown by theability to form cells representing the three germ layers.

Example 3 Production of SOX17 Antibody

A primary obstacle to the identification of definitive endoderm in hESCcultures is the lack of appropriate tools. We therefore undertook theproduction of an antibody raised against human SOX17 protein.

The marker SOX17 is expressed throughout the definitive endoderm as itforms during gastrulation and its expression is maintained in the guttube (although levels of expression vary along the A-P axis) untilaround the onset of organogenesis. SOX17 is also expressed in a subsetof extra-embryonic endoderm cells. No expression of this protein hasbeen observed in mesoderm or ectoderm. It has now been discovered thatSOX17 is an appropriate marker for the definitive endoderm lineage whenused in conjunction with markers to exclude extra-embryonic lineages.

As described in detail herein, the SOX17 antibody was utilized tospecifically examine effects of various treatments and differentiationprocedures aimed at the production of SOX17 positive definitive endodermcells. Other antibodies reactive to AFP, SPARC and Thrombomodulin werealso employed to rule out the production of visceral and parietalendoderm (extra-embryonic endoderm).

In order to produce an antibody against SOX17, a portion of the humanSOX17 cDNA (SEQ ID NO: 1) corresponding to amino acids 172-414 (SEQ IDNO: 2) in the carboxyterminal end of the SOX17 protein (FIG. 2) was usedfor genetic immunization in rats at the antibody production company,GENOVAC (Freiberg, Germany), according to procedures developed there.Procedures for genetic immunization can be found in U.S. Pat. Nos.5,830,876, 5,817,637, 6,165,993 and 6,261,281 as well as InternationalPatent Application Publication Nos. WO00/29442 and WO99/13915, thedisclosures of which are incorporated herein by reference in theirentireties.

Other suitable methods for genetic immunization are also described inthe non-patent literature. For example, Barry et al. describe theproduction of monoclonal antibodies by genetic immunization inBiotechniques 16: 616-620, 1994, the disclosure of which is incorporatedherein by reference in its entirety. Specific examples of geneticimmunization methods to produce antibodies against specific proteins canbe found, for example, in Costaglia et al., (1998) Genetic immunizationagainst the human thyrotropin receptor causes thyroiditis and allowsproduction of monoclonal antibodies recognizing the native receptor, J.Immunol. 160: 1458-1465; Kilpatrick et al (1998) Gene gun deliveredDNA-based immunizations mediate rapid production of murine monoclonalantibodies to the Flt-3 receptor, Hybridoma 17: 569-576; Schmolke etal., (1998) Identification of hepatitis G virus particles in human serumby E2-specific monoclonal antibodies generated by DNA immunization, J.Virol. 72: 4541-4545; Krasemann et al., (1999) Generation of monoclonalantibodies against proteins with an unconventional nucleic acid-basedimmunization strategy, J. Biotechnol 73: 119-129; and Ulivieri et al.,(1996) Generation of a monoclonal antibody to a defined portion of theHeliobacter pylori vacuolating cytotoxin by DNA immunization, J.Biotechnol. 51: 191-194, the disclosures of which are incorporatedherein by reference in their entireties.

SOX7 and SOX18 are the closest Sox family relatives to SOX17 as depictedin the relational dendrogram shown in FIG. 3. We employed the human SOX7polypeptide as a negative control to demonstrate that the SOX17 antibodyproduced by genetic immunization is specific for SOX17 and does notreact with its closest family member. In particular, SOX7 and otherproteins were expressed in human fibroblasts, and then, analyzed forcross reactivity with the SOX17 antibody by Western blot and ICC. Forexample, the following methods were utilized for the production of theSOX17, SOX7 and EGFP expression vectors, their transfection into humanfibroblasts and analysis by Western blot. Expression vectors employedfor the production of SOX17, SOX7, and EGFP were pCMV6 (OriGeneTechnologies, Inc., Rockville, Md.), pCMV-SPORT6 (Invitrogen, Carlsbad,Calif.) and pEGFP-N1 (Clonetech, Palo Alto, Calif.), respectively. Forprotein production, telomerase immortalized MX human fibroblasts weretransiently transfected with supercoiled DNA in the presence ofLipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Total cellularlysates were collected 36 hours post-transfection in 50 mM TRIS-HCl (pH8), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, containing a cocktail ofprotease inhibitors (Roche Diagnostics Corporation, Indianapolis, Ind).Western blot analysis of 100 μg of cellular proteins, separated bySDS-PAGE on NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad,Calif.), and transferred by electro-blotting onto PDVF membranes(Hercules, Calif.), were probed with a 1/1000 dilution of the rat SOX17anti-serum in 10 mM TRIS-HCl (pH 8), 150 mM NaCl 10% BSA, 0.05% Tween-20(Sigma, St. Louis, Mo.), followed by Alkaline Phosphatase conjugatedanti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.), andrevealed through Vector Black Alkaline Phosphatase staining (VectorLaboratories, Burlingame, Calif.). The proteins size standard used waswide range color markers (Sigma, St. Louis, Mo.).

In FIG. 4, protein extracts made from human fibroblast cells that weretransiently transfected with SOX17, SOX7 or EGFP eDNA's were probed onWestern blots with the SOX17 antibody. Only the protein extract fromhSOX17 transfected cells produced a band of ˜51 Kda which closelymatched the predicted 46 Kda molecular weight of the human SOX17protein. There was no reactivity of the SOX17 antibody to extracts madefrom either human SOX7 or EGFP transfected cells. Furthermore, the SOX17antibody clearly labeled the nuclei of human fibroblast cellstransfected with the hSOX17 expression construct but did not label cellstransfected with EGFP alone. As such, the SOX17 antibody exhibitsspecificity by ICC.

Example 4 Validation of SOX17 Antibody as a Marker of DefinitiveEndoderm

Partially differentiated hESCs were co-labeled with SOX17 and AFPantibodies to demonstrate that the SOX17 antibody is specific for humanSOX17 protein and furthermore marks definitive endoderm. It has beendemonstrated that SOX17, SOX7 (which is a closely related member of theSOX gene family subgroup F (FIG. 3)) and AFP are each expressed invisceral endoderm. However, AFP and SOX7 are not expressed in definitiveendoderm cells at levels detectable by ICC, and thus, they can beemployed as negative markers for bonifide definitive endoderm cells. Itwas shown that SOX17 antibody labels populations of cells that exist asdiscrete groupings of cells or are intermingled with AFP positive cells.In particular, FIG. 5A shows that small numbers of SOX17 cells wereco-labeled with AFP; however, regions were also found where there werelittle or no AFP⁺ cells in the field of SOX17⁺ cells (FIG. 5B).Similarly, since parietal endoderm has been reported to express SOX17,antibody co-labeling with SOX17 together with the parietal markers SPARCand/or Thrombomodulin (TM) can be used to identify the SOX17⁺ cells thatare parietal endoderm. As shown in FIGS. 6A-C, Thrombomodulin and SOX17co-labeled parietal endoderm cells were produced by randomdifferentiation of hES cells.

In view of the above cell labeling experiments, the identity of adefinitive endoderm cell can be established by the marker profileSOX17^(hi)/AFP^(lo)/[TM^(lo) or SPARC^(lo)]. In other words, theexpression of the SOX17 marker is greater than the expression of the AFPmarker, which is characteristic of visceral endoderm, and the TM orSPARC markers, which are characteristic of parietal endoderm.Accordingly, those cells positive for SOX17 but negative for AFP andnegative for TM or SPARC are definitive endoderm.

As a further evidence of the specificity of theSOX17^(hi)/AFP^(lo)/TM^(lo)/SPARC^(lo) marker profile as predictive ofdefinitive endoderm, SOX17 and AFP gene expression was quantitativelycompared to the relative number of antibody labeled cells. As shown inFIG. 7A, hESCs treated with retinoic acid (visceral endoderm inducer),or activin A (definitive endoderm inducer), resulted in a 10-folddifference in the level of SOX17 mRNA expression. This result mirroredthe 10-fold difference in SOX17 antibody-labeled cell number (FIG. 7B).Furthermore, as shown in FIG. 8A, activin A treatment of hESCssuppressed AFP gene expression by 6.8-fold in comparison to notreatment. This was visually reflected by a dramatic decrease in thenumber of AFP labeled cells in these cultures as shown in FIGS. 8B-C. Toquantify this further it was demonstrated that this approximately 7-folddecrease in AFP gene expression was the result of a similar 7-folddecrease in AFP antibody-labeled cell number as measured by flowcytometry (FIGS. 9A-B). This result is extremely significant in that itindicates that quantitative changes in gene expression as seen by Q-PCRmirror changes in cell type specification as observed by antibodystaining.

Incubation of hESCs in the presence of Nodal family members (Nodal,activin A and activin B-NAA) resulted in a significant increase in SOX17antibody-labeled cells over time. By 5 days of continuous activintreatment greater than 50% of the cells were labeled with SOX17 (FIGS.10A-F). There were few or no cells labeled with AFP after 5 days ofactivin treatment.

In summary, the antibody produced against the carboxy-terminal 242 aminoacids of the human SOX17 protein identified human SOX17 protein onWestern blots but did not recognize SOX7, it's closest Sox familyrelative. The SOX17 antibody recognized a subset of cells indifferentiating hESC cultures that were primarily SOX17⁺/AFP^(lo/−)(greater than 95% of labeled cells) as well as a small percentage (<5%)of cells that co-label for SOX17 and AFP (visceral endoderm). Treatmentof hESC cultures with activins resulted in a marked elevation of SOX17gene expression as well as SOX17 labeled cells and dramaticallysuppressed the expression of AFP mRNA and the number of cells labeledwith AFP antibody.

Example 5 Q-PCR Gene Expression Assay

In the following experiments, real-time quantitative RT-PCR (Q-PCR) wasthe primary assay used for screening the effects of various treatmentson hESC differentiation. In particular, real-time measurements of geneexpression were analyzed for multiple marker genes at multiple timepoints by Q-PCR. Marker genes characteristic of the desired as well asundesired cell types were evaluated to gain a better understanding ofthe overall dynamics of the cellular populations. The strength of Q-PCRanalysis includes its extreme sensitivity and relative ease ofdeveloping the necessary markers, as the genome sequence is readilyavailable. Furthermore, the extremely high sensitivity of Q-PCR permitsdetection of gene expression from a relatively small number of cellswithin a much larger population. In addition, the ability to detect verylow levels of gene expression provides indications for “differentiationbias” within the population. The bias towards a particulardifferentiation pathway, prior to the overt differentiation of thosecellular phenotypes, is unrecognizable using immunocytochemicaltechniques. For this reason, Q-PCR provides a method of analysis that isat least complementary and potentially much superior toimmunocytochemical techniques for screening the success ofdifferentiation treatments. Additionally, Q-PCR provides a mechanism bywhich to evaluate the success of a differentiation protocol in aquantitative format at semi-high throughput scales of analysis.

The approach taken here was to perform relative quantitation using SYBRGreen chemistry on a Rotor Gene 3000 instrument (Corbett Research) and atwo-step RT-PCR format. Such an approach allowed for the banking of cDNAsamples for analysis of additional marker genes in the future, thusavoiding variability in the reverse transcription efficiency betweensamples.

Primers were designed to lie over exon-exon boundaries or span intronsof at least 800 bp when possible, as this has been empiricallydetermined to eliminate amplification from contaminating genomic DNA.When marker genes were employed that do not contain introns or theypossess pseudogenes, DNase I treatment of RNA samples was performed.

We routinely used Q-PCR to measure the gene expression of multiplemarkers of target and non-target cell types in order to provide a broadprofile description of gene expression in cell samples. The markersrelevant for the early phases of hESC differentiation (specificallyectoderm, mesoderm, definitive endoderm and extra-embryonic endoderm)and for which validated primer sets are available are provided below inTable 1. The human specificity of these primer sets has also beendemonstrated. This is an important fact since the hESCs were often grownon mouse feeder layers. Most typically, triplicate samples were takenfor each condition and independently analyzed in duplicate to assess thebiological variability associated with each quantitative determination.

To generate PCR template, total RNA was isolated using RNeasy (Qiagen)and quantitated using RiboGreen (Molecular Probes). Reversetranscription from 350-500 ng of total RNA was carried out using theiScript reverse transcriptase kit (BioRad), which contains a mix ofoligo-dT and random primers. Each 20 μL reaction was subsequentlydiluted up to 100 μL total volume and 3 μL was used in each 10 μL Q-PCRreaction containing 400 nM forward and reverse primers and 5 μL 2× SYBRGreen master mix (Qiagen). Two step cycling parameters were usedemploying a 5 second denature at 85-94° C. (specifically selectedaccording to the melting temp of the amplicon for each primer set)followed by a 45 second anneal/extend at 60° C. Fluorescence data wascollected during the last 15 seconds of each extension phase. A threepoint, 10-fold dilution series was used to generate the standard curvefor each run and cycle thresholds (Ct's) were converted to quantitativevalues based on this standard curve. The quantitated values for eachsample were normalized to housekeeping gene performance and then averageand standard deviations were calculated for triplicate samples. At theconclusion of PCR cycling, a melt curve analysis was performed toascertain the specificity of the reaction. A single specific product wasindicated by a single peak at the T_(m) appropriate for that PCRamplicon. In addition, reactions performed without reverse transcriptaseserved as the negative control and do not amplify.

A first step in establishing the Q-PCR methodology was validation ofappropriate housekeeping genes (HGs) in the experimental system. Sincethe HG was used to normalize across samples for the RNA input, RNAintegrity and RT efficiency, it was of value that the HG exhibited aconstant level of expression over time in all sample types in order forthe normalization to be meaningful. We measured the expression levels ofCyclophilin G, hypoxanthine phosphoribosyltransferase 1 (HPRT),beta-2-microglobulin, hydroxymethylbiane synthase (HMBS), TATA-bindingprotein CTBP), and glucoronidase beta (GUS) in differentiating hESCs.Our results indicated that beta-2-microglobulin expression levelsincreased over the course of differentiation and therefore we excludedthe use of this gene for normalization. The other genes exhibitedconsistent expression levels over time as well as across treatments. Weroutinely used both Cyclophilin G and GUS to calculate a normalizationfactor for all samples. The use of multiple HGs simultaneously reducesthe variability inherent to the normalization process and increases thereliability of the relative gene expression values.

After obtaining genes for use in normalization, Q-PCR was then utilizedto determine the relative gene expression levels of many marker genesacross samples receiving different experimental treatments. The markergenes employed have been chosen because they exhibit enrichment inspecific populations representative of the early germ layers and inparticular have focused on sets of genes that are differentiallyexpressed in definitive endoderm and extra-embryonic endoderm. Thesegenes as well as their relative enrichment profiles are highlighted inTable 1.

TABLE 1 Germ Layer Gene Expression Domains Endoderm SOX17 definitive,visceral and parietal endoderm MIXL1 endoderm and mesoderm GATA4definitive and primitive endoderm HNF3b definitive endoderm andprimitive endoderm, mesoderm, neural plate GSC endoderm and mesodermExtra- SOX7 visceral endoderm embryonic AFP visceral endoderm, liverSPARC parietal endoderm TM parietal endoderm/trophectoderm Ectoderm ZIC1neural tube, neural progenitors Mesoderm BRACH nascent mesoderm

Since many genes are expressed in more than one germ layer it is usefulto quantitatively compare expression levels of many genes within thesame experiment. SOX17 is expressed in definitive endoderm and to asmaller extent in visceral and parietal endoderm. SOX7 and AFP areexpressed in visceral endoderm at this early developmental time point.SPARC and TM are expressed in parietal endoderm and Brachyury isexpressed in early mesoderm.

Definitive endoderm cells were predicted to express high levels of SOX17mRNA and low levels of AFP and SOX7 (visceral endoderm), SPARC (parietalendoderm) and Brachyury (mesoderm). In addition, ZIC1 was used here tofurther rule out induction of early ectoderm. Finally, GATA4 and HNF3bwere expressed in both definitive and extra-embryonic endoderm, andthus, correlate with SOX17 expression in definitive endoderm (Table 1).A representative experiment is shown in FIGS. 11-14 which demonstrateshow the marker genes described in Table 1 correlate with each otheramong the various samples, thus highlighting specific patterns ofdifferentiation to definitive endoderm and extra-embryonic endoderm aswell as to mesodermal and neural cell types.

In view of the above data it is clear that increasing doses of activinresulted in increasing SOX17 gene expression. Further this SOX17expression predominantly represented definitive endoderm as opposed toextra-embryonic endoderm. This conclusion stems from the observationthat SOX17 gene expression was inversely correlated with AFP, SOX7, andSPARC gene expression.

Example 6 Directed Differentiation of Human ES Cells to DefinitiveEndoderm

Human ES cell cultures randomly differentiate if cultured underconditions that do not actively maintain their undifferentiated state.This heterogeneous differentiation results in production ofextra-embryonic endoderm cells comprised of both parietal and visceralendoderm (APP, SPARC and SOX7 expression) as well as early ectodermaland mesodermal derivatives as marked by ZIC1 and Nestin (ectoderm) andBrachyury (mesoderm) expression. Definitive endoderm cell appearance hasnot been examined or specified for lack of specific antibody markers inES cell cultures. As such, and by default, early definitive endodermproduction in ES cell cultures has not been well studied. Sincesatisfactory antibody reagents for definitive endoderm cells have beenunavailable, most of the characterization has focused on ectoderm andextra-embryonic endoderm. Overall, there are significantly greaternumbers of extra-embryonic and neurectodermal cell types in comparisonto SOX17^(hi) definitive endoderm cells in randomly differentiated EScell cultures.

As undifferentiated hESC colonies expand on a bed of fibroblast feeders,the cells at the edges of the colony take on alternative morphologiesthat are distinct from those cells residing within the interior of thecolony. Many of these outer edge cells can be distinguished by theirless uniform, larger cell body morphology and by the expression ofhigher levels of OCT4. It has been described that as ES cells begin todifferentiate they alter the levels of OCT4 expression up or downrelative to undifferentiated ES cells. Alteration of OCT4 levels aboveor below the undifferentiated threshold may signify the initial stagesof differentiation away from the pluripotent state.

When undifferentiated colonies were examined by SOX17immunocytochemistry, occasionally small 10-15-cell clusters ofSOX17-positive cells were detected at random locations on the peripheryand at the junctions between undifferentiated hESC colonies. As notedabove, these scattered pockets of outer colony edges appeared to be someof the first cells to differentiate away from the classical ES cellmorphology as the colony expanded in size and became more crowded.Younger, smaller fully undifferentiated colonies (<1 mm; 4-5 days old)showed no SOX17 positive cells within or at the edges of the colonieswhile older, larger colonies (1-2 mm diameter, >5days old) had sporadicisolated patches of SOX17 positive, AFP negative cells at the peripheryof some colonies or in regions interior to the edge that did not displaythe classical HESC morphology described previously. Given that this wasthe first development of an effective SOX17 antibody, definitiveendoderm cells generated in such early “undifferentiated” ES cellcultures have never been previously demonstrated.

Based on negative correlations of SOX17 and SPARC gene expression levelsby Q-PCR, the vast majority of these SOX17 positive, AFP negative cellswill be negative for parietal endoderm markers by antibody co-labeling.This was specifically demonstrated for TM-expressing parietal endodermcells as shown in FIGS. 15A-B. Exposure to Nodal factors activin A and Bresulted in a dramatic decrease in the intensity of TM expression andthe number of TM positive cells. By triple labeling using SOX17, AFP andTM antibodies on an activin treated culture, clusters of SOX17 positivecells that were also negative for AFP and TM were observed (FIGS.16A-D). These are the first cellular demonstrations of SOX17 positivedefinitive endoderm cells in differentiating hESC cultures (FIGS. 16A-Dand 17).

With the SOX17 antibody and Q-PCR tools described above we have exploreda number of procedures capable of efficiently programming hESCs tobecome SOX17^(hi)/AFP^(lo)/SPARC/TM^(lo) definitive endoderm cells. Weapplied a variety of differentiation protocols aimed at increasing thenumber and proliferative capacity of these cells as measured at thepopulation level by Q-PCR for SOX17 gene expression and at the level ofindividual cells by antibody labeling of SOX17 protein.

We were the first to analyze and describe the effect of TGFβ familygrowth factors, such as Nodallactivin/BMP, for use in creatingdefinitive endoderm cells from embryonic stem cells in in vitro cellcultures. In typical experiments, activin A, activin B, BMP orcombinations of these growth factors were added to cultures ofundifferentiated human stem cell line hESCyt-25 to begin thedifferentiation process.

As shown in FIG. 19, addition of activin A at 100 ng/ml resulted in a19-fold induction of SOX17 gene expression vs. undifferentiated hESCs byday 4 of differentiation. Adding activin B, a second member of theactivin family, together with activin A, resulted in a 37-fold inductionover undifferentiated hESCs by day 4 of combined activin treatment.Finally, adding a third member of the TGFβ family from the Nodal/Activinand BMP subgroups, BMP4, together with activin A and activin B,increased the fold induction to 57 times that of undifferentiated hESCs(FIG. 19). When SOX17 induction with activins and BMP was compared to nofactor medium controls 5-, 10-, and 15-fold inductions resulted at the4-day time point. By five days of triple treatment with activins A, Band BMP, SOX17 was induced more than 70 times higher than hESCs. Thesedata indicate that higher doses and longer treatment times of theNodal/activin TGFβ family members results in increased expression ofSOX17.

Nodal and related molecules activin A, B and BMP facilitate theexpression of SOX17 and definitive endoderm formation in vivo or invitro. Furthermore, addition of BMP results in an improved SOX17induction possibly through the further induction of Cripto, the Nodalco-receptor.

We have demonstrated that the combination of activins A and B togetherwith BMP4 result in additive increases in SOX17 induction and hencedefinitive endoderm formation. BMP4 addition for prolonged periods (>4days), in combination with activin A and B may induce SOX17 in parietaland visceral endoderm as well as definitive endoderm. In someembodiments of the present invention, it is therefore valuable to removeBMP4 from the treatment within 4 days of addition.

To determine the effect of TGFβ factor treatment at the individual celllevel, a time course of TGFβ factor addition was examined using SOX17antibody labeling. As previously shown in FIGS. 10A-F, there was adramatic increase in the relative number of SOX17 labeled cells overtime. The relative quantification (FIG. 20) shows more than a 20-foldincrease in SOX17-labeled cells. This result indicates that both thenumbers of cells as well SOX17 gene expression level are increasing withtime of TGFβ factor exposure. As shown in FIG. 21, after four days ofexposure to Nodal, activin A, activin B and BMP4, the level of SOX17induction reached 168-fold over undifferentiated hESCs. FIG. 22 showsthat the relative number of SOX17-positive cells was also doseresponsive activin A doses of 100 ng/ml or more were capable of potentlyinducing SOX17 gene expression and cell number.

In addition to the TGFβ family members, the Wnt family of molecules mayplay a role in specification and/or maintenance of definitive endoderm.The use of Wnt molecules was also beneficial for the differentiation ofhESCs to definitive endoderm as indicated by the increased SOX17 geneexpression in samples that were treated with activins plus Wnt3a overthat of activins alone (FIG. 23).

All of the experiments described above were performed using a tissueculture medium containing 10% serum with added factors. Surprisingly, wediscovered that the concentration of serum had an effect on the level ofSOX17 expression in the presence of added activins as shown in FIGS.24A-C. When serum levels were reduced from 10% to 2%, SOX17 expressiontripled in the presence of activins A and B.

Finally, we demonstrated that activin induced SOX17⁺ cells divide inculture as depicted in FIGS. 25A-D. The arrows show cells labeled withSOX17/PCNA/DAPI that are in mitosis as evidenced by thePCNA/DAPI-labeled mitotic plate pattern and the phase contrast mitoticprofile.

Example 7 Chemokine Receptor 4 (CXCR4) Expression Correlates withMarkers for Definitive Endoderm and not Markers for Eesoderm, Ectodermor Visceral Endoderm

As described above, hESCs can be induced to differentiate to thedefinitive endoderm germ layer by the application of cytokines of theTGFβ family and more specifically of the activin/nodal subfamily.Additionally, we have shown that the proportion of fetal bovine serum(FBS) in the differentiation culture medium effects the efficiency ofdefinitive endoderm differentiation from hESCs. This effect is such thatat a given concentration of activin A in the medium, higher levels ofFBS will inhibit maximal differentiation to definitive endoderm. In theabsence of exogenous activin A, differentiation of hESCs to thedefinitive endoderm lineage is very inefficient and the FBSconcentration has much milder effects on the differentiation process ofhESCs.

In these experiments, hESCs were differentiated by growing in RPMImedium (Invitrogen, Carlsbad, Calif.; cat #61870-036) supplemented with0.5%, 2.0% or 10% FBS and either with or without 100 ng/ml activin A for6 days. In addition, a gradient of FBS ranging from 0.5% to 2.0% overthe first three days of differentiation was also used in conjunctionwith 100 ng/ml of activin A. After the 6 days, replicate samples werecollected from each culture condition and analyzed for relative geneexpression by real-time quantitative PCR. The remaining cells were fixedfor immunofluorescent detection of SOX17 protein.

The expression levels of CXCR4 varied dramatically across the 7 cultureconditions used (FIG. 26). In general, CXCR4 expression was high inactivin A treated cultures (A100) and low in those which did not receiveexogenous activin A (NF). In addition, among the A100 treated cultures,CXCR4 expression was highest when FBS concentration was lowest. Therewas a remarkable decrease in CXCR4 level in the 10% FBS condition suchthat the relative expression was more in line with the conditions thatdid not receive activin A (NF).

As described above, expression of the SOX17, GSC, MIXL1, and HNF3β genesis consistent with the characterization of a cell as definitiveendoderm. The relative expression of these four genes across the 7differentiation conditions mirrors that of CXCR4 (FIGS. 27A-D). Thisdemonstrates that CXCR4 is also a marker of definitive endoderm.

Ectoderm and mesoderm lineages can be distinguished from definitiveendoderm by their expression of various markers. Early mesodermexpresses the genes Brachyury and MOX1 while nascent neuro-ectodermexpresses SOX1 and ZIC1. FIGS. 28A-D demonstrate that the cultures whichdid not receive exogenous activin A were preferentially enriched formesoderm and ectoderm gene expression and that among the activin Atreated cultures, the 10% FBS condition also had increased levels ofmesoderm and ectoderm marker expression. These patterns of expressionwere inverse to that of CXCR4 and indicated that CXCR4 was not highlyexpressed in mesoderm or ectoderm derived from hESCs at thisdevelopmental time period.

Early during mammalian development, differentiation to extra-embryoniclineages also occurs. Of particular relevance here is thedifferentiation of visceral endoderm that shares the expression of manygenes in common with definitive endoderm, including SOX17. Todistinguish definitive endoderm from extra-embryonic visceral endodermone should examine a marker that is distinct between these two. SOX7represents a marker that is expressed in the visceral endoderm but notin the definitive endoderm lineage. Thus, culture conditions thatexhibit robust SOX17 gene expression in the absence of SOX7 expressionare likely to contain definitive and not visceral endoderm. It is shownin FIG. 28E that SOX7 was highly expressed in cultures that did notreceive activin A, SOX7 also exhibited increased expression even in thepresence of activin A when FBS was included at 10%. This pattern is theinverse of the CXCR4 expression pattern and suggests that CXCR4 is nothighly expressed in visceral endoderm.

The relative number of SOX17 immunoreactive (SOX17) cells present ineach of the differentiation conditions mentioned above was alsodetermined. When hESCs were differentiated in the presence of high doseactivin A and low FBS concentration (0.5% -2.0%) SOX17⁺ cells wereubiquitously distributed throughout the culture. When high dose activinA was used but FBS was included at 10% (v/v), the SOX17⁺ cells appearedat much lower frequency and always appeared in isolated clusters ratherthan evenly distributed throughout the culture (FIGS. 29A and C as wellas B and E). A further decrease in SOX17⁺ cells was seen when noexogenous activin A was used. Under these conditions the SOX17⁺ cellsalso appeared in clusters and these clusters were smaller and much morerare than those found in the high activin A, low FBS treatment (FIGS.29C and F). These results demonstrate that the CXCR4 expression patternsnot only correspond to definitive endoderm gene expression but also tothe number of definitive endoderm cells in each condition.

Example 8 Differentiation Conditions that Enrich for Definitive EndodermIncrease the Proportion of CXCR4 Positive Cells

The dose of activin A also effects the efficiency at which definitiveendoderm can be derived from hESCs. This example demonstrates thatincreasing the dose of activin A increases the proportion of CXCR4⁺cells in the culture.

hESCs were differentiated in RPMI media supplemented with 0.5%-2% FBS(increased from 0.5% to 1.0% to 2.0% over the first 3 days ofdifferentiation) and either 0, 10, or 100 ng/ml of activin A. After 7days of differentiation the cells were dissociated in PBS withoutCa²⁺/Mg²⁺ containing 2% FBS and 2 mM (EDTA) for 5 minutes at roomtemperature. The cells were filtered through 35 μm nylon filters,counted and pelleted. Pellets were resuspended in a small volume of 50%human serum/50% normal donkey serum and incubated for 2 minutes on iceto block non-specific antibody binding sites. To this, 1 μl of mouseanti-CXCR4 antibody (Abcam, cat #ab10403-100) was added per 50 μl(containing approximately 10⁵ cells) and labeling proceeded for 45minutes on ice. Cells were washed by adding 5 ml of PBS containing 2%human serum (buffer) and pelleted. A second wash with 5 ml of buffer wascompleted then cells were resuspended in 50 μl buffer per 10⁵ cells.Secondary antibody (FITC conjugated donkey anti-mouse; JacksonImmunoResearch, cat #715-096-151) was added at 5 μg/ml finalconcentration and allowed to label for 30 minutes followed by two washesin buffer as above. Cells were resuspended at 5×10⁶ cells/ml in bufferand analyzed and sorted using a FACS Vantage (Beckton Dickenson) by thestaff at the flow cytometry core facility (The Scripps ResearchInstitute). Cells were collected directly into RLT lysis buffer (Qiagen)for subsequent isolation of total RNA for gene expression analysis byreal-time quantitative PCR.

The number of CXCR4⁺ cells as determined by flow cytometry were observedto increase dramatically as the dose of activin A was increased in thedifferentiation culture media FIGS. 30A-C). The CXCR4⁺ cells were thosefalling within the R4 gate and this gate was set using a secondaryantibody-only control for which 0.2% of events were located in the R4gate. The dramatically increased numbers of CXCR4⁺ cells correlates witha robust increase in definitive endoderm gene expression as activin Adose is increased (FIGS. 31A-D).

Example 9 Isolation of CXCR4 Positive Cells Enriches for DefinitiveEndoderm Gene Expression and Depletes Cells Expressing Markers ofMesoderm, Ectoderm and Visceral Endoderm

The CXCR4⁺ and CXCR4⁻ cells identified in Example 8 above were collectedand analyzed for relative gene expression and the gene expression of theparent populations was determined simultaneously.

The relative levels of CXCR4 gene expression was dramatically increasedwith increasing dose of activin A (FIG. 32). This correlated very wellwith the activin A dose-dependent increase of CXCR4⁺ cells (FIGS.30A-C). It is also clear that isolation of the CXCR4⁺ cells from eachpopulation accounted for nearly all of the CXCR4 gene expression in thatpopulation. This demonstrates the efficiency of the FACS method forcollecting these cells.

Gene expression analysis revealed that the CXCR4⁺ cells contain not onlythe majority of the CXCR4 gene expression, but they also contained geneexpression for other markers of definitive endoderm. As shown in FIGS.31A-D, the CXCR4⁺ cells were further enriched over the parent A100population for SOX17, GSC, HNF3B, and MIXL1. In addition, the CXCR4⁻fraction contained very little gene expression for these definitiveendoderm markers. Moreover, the CXCR4⁺ and CXCR4⁻ populations displayedthe inverse pattern of gene expression for markers of mesoderm, ectodermand extra-embryonic endoderm. FIGS. 33A-D shows that the CXCR4⁺ cellswere depleted for gene expression of Brachyury, MOX1, ZIC1, and SOX7relative to the A100 parent population. This A100 parent population wasalready low in expression of these markers relative to the low dose orno activin A conditions. These results show that the isolation of CXCR4⁺cells from hESCs differentiated in the presence of high activin A yieldsa population that is highly enriched for and substantially puredefinitive endoderm.

Example 10 Quantitation of Definitive Endoderm Cells in a CellPopulation using CXCR4

To confirm the quantitation of the proportion of definitive endodermcells present in a cell culture or cell population as determinedpreviously herein and as determined in U.S. Provisional PatentApplication No. 60/532,004, entitled DEFINITIVE ENDODERM, filed Dec. 23,2003, the disclosure of which is incorporated herein by reference in itsentirety, cells expressing CXCR4 and other markers of definitiveendoderm were analyzed by FACS.

Using the methods such as those described in the above Examples, hESCswere differentiated to produce definitive endoderm. In particular, toincrease the yield and purity in differentiating cell cultures, theserum concentration of the medium was controlled as follows: 0.2% FBS ondayl, 1.0% FBS on day 2 and 2.0% FBS on days 3-6. Differentiatedcultures were sorted by FACS using three cell surface epitopes,E-Cadherin, CXCR4, and Thrombomodulin. Sorted cell populations were thenanalyzed by Q-PCR to determine relative expression levels of markers fordefinitive and extraembryonic-endoderm as well as other cell types.CXCR4 sorted cells taken from optimally differentiated cultures resultedin the isolation of definitive endoderm cells that were >98% pure.

Table 2 shows the results of a marker analysis for a definitive endodermculture that was differentiated from hESCs using the methods describedherein.

TABLE 2 Composition of Definitive Endoderm Cultures Percent PercentPercent Percent of Definitive Extraembryonic hES Marker(s) cultureEndoderm endoderm cells SOX17 70-80 100 Thrombomodulin <2 0 75 AFP <1 025 CXCR4 70-80 100 0 ECAD 10 0 100 other (ECAD neg.) 10-20 Total 100 100 100 100

In particular, Table 2 indicates that CXCR4 and SOX17 positive cells(endoderm) comprised from 70%-80% of the cells in the cell culture. Ofthese SOX17-expressing cells, less than 2% expressed TM (parietalendoderm) and less than 1% expressed AFP (visceral endoderm). Aftersubtracting the proportion of TM-positive and AFP-positive cells(combined parietal and visceral endoderm; 3% total) from the proportionof SOX17/CXCR4 positive cells, it can be seen that about 67% to about77% of the cell culture was definitive endoderm. Approximately 10% ofthe cells were positive for E-Cadherin (ECAD), which is a marker forhESCs, and about 10-20% of the cells were of other cell types.

We have discovered that the purity of definitive endoderm in thedifferentiating cell cultures that are obtained prior to FACS separationcan be improved as compared to the above-described low serum procedureby maintaining the FBS concentration at <0.5% throughout the 5-6 daydifferentiation procedure. However, maintaining the cell culture at≦0.5% throughout the 5-6 day differentiation procedure also results in areduced number of total definitive endoderm cells that are produced.

Definitive endoderm cells produced by methods described herein have beenmaintained and expanded in culture in the presence of activin forgreater than 50 days without appreciable differentiation. In such cases,SOX17, CXCR4, MIXL1, GATA4, HNF3β expression is maintained over theculture period. Additionally, TM, SPARC, OCT4, AFP, SOX7, ZIC1 and BRACHwere not detected in these cultures. It is likely that such cells can bemaintained and expanded in culture for substantially longer than 50 dayswithout appreciable differentiation.

Example 11 Additional Marker of Definitive Endoderm Cells

In the following experiment, RNA was isolated from purified definitiveendoderm and human embryonic stem cell populations. Gene expression wasthen analyzed by gene chip analysis of the RNA from each purifiedpopulation. Q-PCR was also performed to further investigate thepotential of genes expressed in definitive endoderm, but not inembryonic stem cells, as a marker for definitive endoderm.

Human embryonic stem cells (hESCs) were maintained in DMEM/F12 mediasupplemented with 20% KnockOut Serum Replacement, 4 ng/ml recombinanthuman basic fibroblast growth factor (bFGF), 0.1 mM 2-mercaptoethanol,L-glutamine, non-essential amino acids and penicillin/streptomycin.hESCs were differentiated to definitive endoderm by culturing for 5 daysin RPMI media supplemented with 100 ng/ml of recombinant human activinA, fetal bovine serum (FBS), and penicillin/streptomycin. Theconcentration of FBS was varied each day as follows: 0.1% (first day),0.2% (second day), 2% (days 3-5).

Cells were isolated by fluorescence activated cell sorting (FACS) inorder to obtain purified populations of hESCs and definitive endodermfor gene expression analysis. Immuno-purification was achieved for hESCsusing SSEA4 antigen (R&D Systems, cat #FAB1435P) and for definitiveendoderm using CXCR4 (R&D Systems, cat #FAB170P). Cells were dissociatedusing trypsin/EDTA (Invitrogen, cat #25300-054), washed in phosphatebuffered saline (PBS) containing 2% human serum and resuspended in 100%human serum on ice for 10 minutes to block non-specific binding.Staining was carried out for 30 minutes on ice by adding 200 μl ofphycoerythrin-conjugated antibody to 5×10⁶ cells in 800 μl human serum.Cells were washed twice with 8 ml of PBS buffer and resuspended in 1 mlof the same. FACS isolation was carried out by the core facility of TheScripps Research Institute using a FACS Vantage (BD Biosciences). Cellswere collected directly into RLT lysis buffer and RNA was isolated byRNeasy according to the manufacturers instructions (Qiagen).

Purified RNA was submitted in duplicate to Expression Analysis (Durham,N.C.) for generation of the expression profile data using the Affymetrixplatform and U133 Plus 2.0 high-density oligonucleotide arrays. Datapresented is a group comparison that identifies genes differentiallyexpressed between the two populations, hESCs and definitive endoderm.Genes that exhibited a robust upward change in expression level overthat found in hESCs were selected as new candidate markers that arehighly characteristic of definitive endoderm. Select genes were assayedby Q-PCR, as described above, to verify the gene expression changesfound on the gene chip and also to investigate the expression pattern ofthese genes during a time course of hESC differentiation.

FIGS. 34A-M show the gene expression results for certain markers.Results are displayed for cell cultures analyzed 1, 3 and 5 days afterthe addition of 100 ng/ml activin A, CXCR4-expressing definitiveendoderm cells purified at the end of the five day differentiationprocedure (CXDE), and in purified hESCs. A comparison of FIGS. 34C andG-M demonstrates that the six marker genes, FGF17, VWF, CALCR, FOXQ1,CMKOR1 and CRIP1, exhibit an expression pattern that is almost identicalto each other and which is also identical to the pattern of expressionof CXCR4 and the ratio of SOX17/SOX7. As described previously, SOX17 isexpressed in both the definitive endoderm as well as in theSOX7-expressing extra-embryonic endoderm. Since SOX7 is not expressed inthe definitive endoderm, the ratio of SOX17/SOX7 provides a reliableestimate of definitive endoderm contribution to the SOX17 expressionwitnessed in the population as a whole. The similarity of panels G-L andM to panel C indicates that FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1are likely markers of definitive endoderm and that they are notsignificantly expressed in extra-embryonic endoderm cells.

It will be appreciated that the Q-PCR results described herein can befurther confirmed by ICC.

Example 12 Generation of SOX17 Promoter-EGFP Transgenic hESC Lines andCXCR4 Promoter-EGFP Transgenic hESC Lines

As an alternative to purification of definitive endoderm using theCXCR4-specific antibody, EGFP fusions to either the SOX17 or the CXCR4promoters can be used. In particular, this Example describes theconstruction of a vector comprising a reporter cassette which comprisesa reporter gene under the control of the SOX17 regulatory region.Additionally, the construction of a vector comprising a reportercassette which comprises a reporter gene under the control of the CXCR4regulatory region is described. This Example also describes thepreparation of a cell, such as a human embryonic stem cell, transfectedwith one or more of these vectors as well as a cell having this one orboth of these reporter cassettes integrated into its genome.

SOX17-expressing definitive endoderm cell lines and CXRC4-expressingdefinitive endoderm cell lines genetically tagged with a reporter geneare constructed by placing a GFP reporter gene under the control of theregulatory region promoter) of the SOX17 gene or the CXCR4 gene,respectively. First, a plasmid construct in which EGFP expression isdriven by the human SOX17 or CXCR4 gene promoter is generated byreplacing the CMV promoter of vector pEGFP-N1 (Clontech) with the humanSOX17 or CXCR4 control region. These control regions contain thecharacterized regulatory elements of either the SOX17 or the CXCR4 gene,and they is sufficient to confer the normal expression pattern of thesegenes in transgenic mice. In the resulting vector, expression of EFGP isdriven by either the SOX17 promoter or the CXCR4 promoter. In someexperiments, this vector can be transfected into hESCs.

The SOX17 promoter/EGFP cassette or the CXCR4 promoter/EGFP cassette isexcised from the above vector, and then subcloned into a selectionvector containing the neomycin phosphotransferase gene under control ofthe phosphoglycerate kinase-1 promoter. The selection cassette isflanked by flp recombinase recognition sites to allow removal of thecassette. This selection vector is linearized, and then introduced intohESCs using standard lipofection methods. Following 10-14 days ofselection in G418, undifferentiated transgenic hESC clones is isolatedand expanded.

It will be appreciated that reporter genes other than GFP or EGFP can beused in any of the above-described constructs provided that the reporterallows for cell separation by FACS

Example 13 Alternative Isolation of Definitive Endoderm

The following Example demonstrates that hESCs comprising a SOX17 orCXCR4 promoter/EGFP cassette can be differentiated into definitiveendoderm cells and then subsequently isolated by fluorescence-activatedcell sorting (FACS).

SOX17 or CXCR4 promoter/EGFP transgenic hESCs are differentiated forapproximately 6, 12 and 18 hours in growth medium containing 100 ng/mlactivin A and no serum. The differentiated cells are then harvested bytrypsin digestion and sorted on a Becton Dickinson FACS Diva directlyinto RNA lysis buffer or PBS. A sample of single live cells is takenwithout gating for EGFP and single live cells are gated into EGFPpositive and GFP negative populations. In a separate experiment, theEGFP positive fraction is separated into two equally sized populationsaccording to fluorescence intensity (Hi and Lo).

Following sorting, cell populations are analyzed by both Q-PCR andimmunocytochemistry. For Q-PCR analysis, RNA is prepared using QiagenRNeasy columns and then converted to cDNA. Q-PCR is conducted asdescribed previously. For immunocytochemistry analysis, cells are sortedinto PBS, fixed for 10 minutes in 4% paraformaldehyde, and adhered toglass slides using a Cytospin centrifuge. The primary antibody SOX17 orCXCR4. An appropriate secondary antibody conjugated to FITC (green) orRhodamine (Red) is used to detect binding of the primary antibody.

Sorted cells are further subjected to Q-PCR analysis. Differentiatedcells show a correlation of EGFP fluorescence with endogenous SOX17 orCXCR4 expression gene expression. Compared to non-fluorescing cells, theEGFP positive cells show a greater than 2-fold increase in SOX17 orCXCR4 expression levels. The separation of high and low EGFP intensitycells indicates that EGFP expression level correlates with SOX17 orCXCR4 expression level. In addition to SOX17 or CXCR4 mRNA analysis,sorted cells are subjected to immunocytochemistry analysis of SOX17 orCXCR4 polypeptide (in embodiments where CXCR4/EGFP fusions are used,SOX17 polypeptide expression is analyzed and in cases where SOX17/EGFPfusions are used, CXCR4 polypeptide expression is analyzed). Substantialexpression of either the SOX17 or CXCR4 polypeptides can be seen in theenriched in the EGFP positive fraction. In contrast, little expressionof either the SOX17 or CXCR4 polypeptides is seen in the EGFP negativefraction.

Given these results, at least about 5% of the cells present in thedifferentiated cell cultures prior to sorting are SOX17/CXCR4-positivedefinitive endoderm cells. At least about 90% of the cells in the sortedcell populations are SOX17/CXCR4-positive definitive endoderm cells.

Example 14 Passage of Definitive Endoderm Cells in Culture

This Example demonstrates that the definitive endoderm cells describedherein can be maintained in cell culture and passaged without furtherdifferentiation.

Definitive endoderm cells were differentiated from two related passages,designated EB and EV, of the CyT25 hESC line in the presence of 100ng/ml activin A in low serum RMPI. The low serum RPMI contained 0%(v/v)fetal bovine serum (FBS) on day 1, 0.2% (v/v) FBS on day two and 2%serum on each day thereafter. After four days of differentiation, thecells maintained in culture in either the presence or absence of 100ng/ml activin A for a total of 36 days as measured from induction ofdifferentiation. During the 36 day culture period, the definitiveendoderm cells were passaged twice. Furthermore, on days 29-36 the cellsof the group designated EV were additionally contacted with 50 mg/mlEGF. On days 4, 9, 23, 29 and 36 of culture, Q-PCR was used to measurethe expression of marker genes indicative of definitive endoderm.

FIGS. 35A-D show that, in cell cultures provided with 100 ng/ml activinA, expression of the definitive endoderm markers SOX17, GSC, MIXL1 andCXCR4 was maintained during the 32 day culture period subsequent to thederivation of the definitive endoderm cells from hESCs (days 4 to 36).Little expression of these markers was observed in the cell culturesgrown in the absence of activin A. Addition of 50 ng/ml EGF did notappear to significantly increase the expression of any of the definitiveendoderm markers.

Example 15 Expansion of Purified Definitive Endoderm Cells

This Example demonstrates that the definitive endoderm cells describedherein can be differentiated from hESCs, purified and then regrown andexpanded in cell culture.

FIG. 36 shows the design of a definitive endoderm purification/expansionexperiment. In particular, definitive endoderm cells were differentiatedfrom the 96^(th) passage of hESC line CyT25 in the presence of 100 ng/mlactivin A in low serum RMPI. The low serum RPMI contained 0%(v/v) fetalbovine serum (FBS) on day 1, 0.2% (v/v) FBS on day two and 2% serum oneach day thereafter. After five days of differentiation, the cells weresubjected to FACS purification using antibody against CXCR4 as describedin previous Examples. The purified cell population was then cultured onIVF dishes coated with poly-ornithine and 10 μg/ml human fibronectin inRPMI containing 2% FBS under one of the following four growth factorconditions: no added factor (NF); 100 ng/ml activin A (A); 100 ng/mlactivin A and 100 ng/ml IGF1 (AI); or 100 ng/ml activin A, 12 ng/ml bFGFand 10 ng/ml EGF (AFE). On day 11, the expanded definitive endodermcells were passaged using the standard trypsinization method. Each ofthe cell cultures were then grown for an additional 10 days afterpassage (a total of 21 days subsequent to the first contact with activinA). Samples of mRNA were obtained at days 0, 5, 11 and 21 as indicatedin FIG. 36.

FIGS. 37A-E show the expression of marker genes for various embryoniccell types at each of the sample time points for each of the cultureconditions. As shown in FIGS. 37A-B, the definitive endoderm markersSOX17 and GSC were highly expressed five day old unpurified definitiveendoderm cultures but were not expressed in hESCs. This expression is incontrast to that of the HESC marker, OCT4 (FIG. 37C). Six days after thepurification of definitive endoderm cells (day 11) the expression ofSOX17 and GSC expression remained high in each of the cell culturestreated with growth factor(s) but not in cell cultures grown in theabsence of activin A (FIG. 37A-B). A similar pattern of expression wasobserved for these markers 10 days after passage (day 21) (FIGS. 37A-B).No expression of mRNA for markers of hESCs (OCT4), mesoderm (brachyury),or ectoderm (ZIC1 and SOX1) was observed in any of the cell culturessubsequent to purification. This result indicates that purifieddefinitive endoderm cells do not form hESCs or cells of the other twoembryonic cell lineages even in the absence of activin A (FIGS. 37C-F).

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

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What is claimed is:
 1. A method of expanding human definitive endodermcells in culture, said method comprising the steps of: (a) providing acell culture comprising at least 20% human definitive endoderm cells,wherein said human definitive endoderm cells are multipotent cells thatcan differentiate into cells of the gut tube or organs derivedtherefrom, and (b) culturing the human definitive endoderm cellsprovided from step (a) under conditions that permit the expansion ofsaid human definitive endoderm cells.
 2. The method of claim 1, whereinthe step of culturing said human definitive endoderm cells comprisespassaging said definitive endoderm cells, thereby producing a pluralityof cell cultures comprising human definitive endoderm cells.
 3. Themethod of claim 2, wherein the step of passaging said human definitiveendoderm cells comprises providing at least one protease to said cellculture.
 4. The method of claim 3, wherein said at least one proteasecomprises trypsin.
 5. The method of claim 2, wherein the step ofpassaging said human definitive endoderm cells comprises mechanicallydisrupting contacts between said human definitive endoderm cells.
 6. Themethod of claim 2, wherein the step of passaging said human definitiveendoderm cells comprises incubating said human definitive endoderm cellsin a cell dispersal buffer.
 7. The method of claim 2, wherein said humandefinitive endoderm cells are attached to a substrate.
 8. The method ofclaim 7, wherein the step of passaging said human definitive endodermcells comprises detaching said human definitive endoderm cells from saidsubstrate.
 9. The method of claim 7, wherein said substrate is a surfaceof a tissue culture flask.
 10. The method of claim 2, wherein the stepof passaging said human definitive endoderm cells comprises providing atleast one enzyme to said cell culture.
 11. The method of claim 1,wherein said human definitive endoderm cells are derived from humanembryonic stem cells (hESCs).